Browsing by Author "Lukey, Pauline"

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    Characterisation of cytolytic CD4+ and suppresor CD8+ activity of T lymphocyte clones derived from tuberculous pleuritis
    (2000) Glashoff, Richard Helmuth; Ress, Stanley; Lukey, Pauline
    Bibliography: leaves 156-174.
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    Human macrophage model for selective evaluation of CD8⁺ and γδ⁺ cytotoxic T cell function in tuberculosis
    (1999) Passmore, Jo-Ann Shelley; Ress, Stanley; Lukey, Pauline; Keraan, Mogamat Mustapha
    The human macrophage cell line U937 was investigated as an in vitro model for human macrophage function in mycobacterial infections. This involved evaluating the ability of differentiated U937 cells to phagocytose Mycobacterium tuberculosis, control intracellular mycobacterial growth, and present mycobacterial antigens to human HLA class I-matched cytotoxic T lymphocytes (CTLs). Differentiation of U937 cells usmg IFN-γ, 1,25-(OH)₂ vitamin D₃, or PMA significantly enhanced their ability to phagocytose M. tuberculosis but failed to induce a subsequent respiratory burst response. Following infection, U937 cells were found to be permissive to the intracellular growth of both the virulent H37Rv strain of M. tuberculosis and the attenuated vaccine strain of M. bovis BCG. U937 cells have been shown to constitutively express high levels of cell surface HLA class I while expressing undetectable levels of HLA class II both at the mRN A level and at the cell surface. HLA class II expression was neither up-regulated following infection with M. tuberculosis nor inducible using IFN-γ, 1,25-(OH)₂ vitamin D₃, PMA, GM-CSF or a combination of these agents. In contrast, chronic infection of U937 cells with virulent H37Rv M. tuberculosis (but not with BCG) resulted in the cell surface expression of HLA class I being significantly up-regulated. Taken together, these characteristics made U937 cells a very attractive model for further investigations into their ability to present mycobacterial antigens to human HLA class I-restricted CTLs. Differentiation of U937 cells was found to completely abrogate their sensitivity to non-antigen specific cytolysis mediated by NK or LAK cells. Following infection with M. tuberculosis, U937 target cells were lysed by M. tuberculosis-primed CTLs from HLA class I-matched donors in an antigen-specific manner and with a similar efficiency to autologous macrophage targets. This cytolytic activity was restricted to live organisms since only U937 cells infected with virulent H37Rv M. tuberculosis and BCG but not those pulsed with soluble PPD were lysed by the HLA class I-matched effector cells. On the other hand, M. tuberculosisstimulated but ALA-mismatched CTLs failed to lyse infected U937 cells in an antigen-specific manner. T cell subset fractionation of the HLA class I-matched M. tuberculosis-primed CTL population and limiting dilution cloning demonstrated that the cytolytic activity was mediated by CD8⁺ cytolytic T cells and confirmed that CD4⁺ T cells showed no significant ability to lyse infected U937 target cells. Furthermore, this study found that M. tuberculosis-infected U937 target cells were lysed by CD8⁺ CTLs more rapidly and strongly than similarly infected autologous macrophage targets demonstrating the sensitivity of this in vitro model as an indicator for CD8⁺ cytolytic function in mycobacterial infections. M. tuberculosis-infected U937 cells were found to be highly sensitive to mycobacterial antigenspecific cytolysis mediated by γδ⁺ CTL. Mycobacterial antigen-specific γδ⁺ CTLs consistently showed stronger cytolytic activity against infected U937 target cells than γδ⁺ CTL but were not restricted to classical HLA class I or class II molecules. A panel of cytolytic human M. tuberculosis-reactive γδ⁺ CTL clones was established to investigate more thoroughly the role of γδ⁺ CTL lytic activity in human mycobacterial infections. This study examined the mechanism of cellular cytotoxicity used by these mycobacterial-specific γδ⁺ CTL clones against infected U93 7 targets and further investigated the effect of γδ⁺ T cell-mediated cytolysis on intracellular mycobacterial survival. Cytolysis mediated by the γδ⁺ T cell clones was found to be dependent on cell-to-cell contact. Furthermore, the ability of the γδ⁺ CTL clones to lyse infected targets was found to be strongly Ca²⁺-dependent, sensitive to cyclosporine A (a specific inhibitor of granule exocytosis ), and completely abrogated following Sr²⁺-induced de-granulation of the γδ⁺ T cell effectors, indicating that cytoxicity was mediated predominantly by the granule exocytosis/ perforin pathway. Despite being strongly cytolytic against infected U937 cells, however, the γδ⁺ CTL clones did not have any impact on the survival of intracellular M. tuberculosis. The major conclusions of this study are that U937 cells not only provide a useful in vitro human macrophage model allowing for selective evaluation of HLA class I-restricted CD8⁺ CTL function in mycobacterial infections but also provided a highly sensitive indicator for γδ⁺ CTL cytolytic activity.
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    Immune parameters as predictors of response to maintenance therapy in low grade non-Hodgkin's lymphoma patients
    (1998) Watkins, Marcia Linda Vivienne; Ress, Stanley; Lukey, Pauline
    The non-Hodgkin's lymphomas (NHL) are a heterogeneous group of malignancies characterised by the uncontrolled proliferation of lymphoid cells. The Working Formulation divides these neoplasms into low, intermediate and high-grade categories according to their natural history. Low grade NHL (LG-NHL) is clinically indolent whereas high grade NHL is more aggressive. Most LG-NHL patients respond well to chemotherapy, but are rarely cured, making death from LG-NHL virtually inevitable. In this study on LG-NHL patients, all patients received combination chemotherapy for 6 weeks, which consisted of cyclophosphamide, oncovin (vincristine) and prednisone (COP). After this treatment patients received either oral cyclophosphamide or alpha-interferon (α-IFN) as maintenance treatment for a period of two years. The ability to predict patients' treatment response at diagnosis would be extremely helpful for both the patient and the clinician. This would enable appropriate therapy to be instituted at diagnosis, whether it be cyclophosphamide or α-IFN, which might increase the subsequent survival of these patients, as this would prevent patients from receiving treatment to which they would not respond. Furthermore, patients who were unlikely to respond to either of these two treatments could be targeted for alternative treatment or form part of a clinical research trial. LG-NHL patients were assessed at diagnosis for a number of immune parameters. These included: - natural killer activity (NKA), α-IFN enhanced NKA, mitogen and antigen proliferation, lymphokine activated kill against both KS62 and Daudi targets, phenotypic analysis of circulating lymphocytes, assessment of IL-2 levels after mitogen stimulation of lymphocytes, as well as the determination of plasma IL-2 levels. All these above-mentioned parameters were serially monitored over time in an attempt to predict early relapse. A statistically significant reduction in percentage of circulating CD3+ and CD8+ cells and an increase in percentage NK cells was found in patients at diagnosis as compared to normal controls. A reduction in percentage of circulating NK cells over time appeared to be a good prognostic indicator and an increase in percentage NK cells a poor prognostic indicator in this group of patients, although this was not statistically significant. When the in vitro α-IFN enhanced NKA was indicated as a percentage of NKA, a negative correlation appeared to exist between this in vitro response to α-IFN and the in vivo response, although this was not statistically significant (i.e. those patients showing the least α-IFN enhanced NKA were those that responded clinically and those with the highest α-IFN augmented NKA either relapsed or transformed to a higher grade of NHL). By incorporating the percentages of circulating lymphocytes present in the peripheral blood, into the multivariate discriminant analysis, it was possible to derive formulae to enable the prediction of response to either α-IFN or cyclophosphamide. This was a particularly exciting and apparently novel finding. The work presented here has therefore established both a possible negative indicator of α-IFN response (a high in vitro α-IFN response) and a positive indicator (the formula generated in the multivariate discriminant analysis using the flow cytometric phenotypic analysis). By making using of both of these factors, it would increase the chances of patients being selected for appropriate forms of treatment and minimise the chances of patients suffering a relapse. The Kaplan-Meier curve indicated that cyclophosphamide treatment was more beneficial than α-IFN therapy in this group of patients, although this did not attain statistical significance. Verification of all of these above-mentioned findings would need further confirmation in a larger study.