Targets for immune mediated killing of tumor cells and T cell functions in B-CLL

University dissertation from Stockholm : Karolinska Institutet, Department of Oncology-Pathology

Abstract: There is a great need for developing and improving treatment alternatives in B-cell chronic lymphocytic leukemia (B-CLL). New therapeutical approaches based on the manipulation of the immune system enable establishment of enhanced control over the malignant clone. Exploitation of molecular defects of leukemic cells causing apoptotic failure and tumor immune escape mechanisms might have impact on a deeper understanding of the pathophysiology of B-CLL and targets for the development of novel therapy modalities of this disease. Analyses of CD95 receptor expression showed a downregulation on the leukemic cells. These B-CLL cells exhibited various functional abnormalities. A weak proliferation of B-CLL cells were induced upon activation with both anti-CD40 and anti-CD95 monoclonal antibodies (mAbs) and stimulation with anti-CD95 mAb did not induce apoptosis. Cross-linking of CD40 and CD95 molecules led to delayed protein tyrosine signals of weak intensities and transient signaling, respectively. Unspecific stimulation of B-CLL cells led to induction of apoptosis, but with a large variation between the different clones. The apoptosis induced was independent on the CD95 receptor expression and the stage of differentiation. Each leukemic clone investigated, upregulated the ex vivo undetectable p53 upon culture with CD40/IL4. No relation between the p53 expression and apoptosis of leukemic cells was noted. There was an inter-individual variation in the expression of Bcl-2 and phosphotyrosine proteins. These proteins were up- or downregulated by both activation manners. The relative expression of Bcl-2 was found to correlate with the degree of apoptosis. Potential target structures for monoclonal antibody therapy i.e. CD20, CD22 and CD52 have been estimated quantitatively by flow cytometry. A considerably lower expression of the CD20 and CD22 antigens was found on leukemic cells compared to control B cells. Furthermore, a substantial variability over the time was seen and most pronounced for CD20. No upregulation of CD20 was observed upon in vivo IL-4 therapy. Highly significant correlations were obtained between antigen receptor numbers on lymphocytes from control donors and B-CLL patients estimated with three different calibrator standards available for quantitative flow cytometry. Furthermore, methodological aspects on the technique are discussed. Estimation of cytokine production in ex vivo T cells revealed higher numbers of IL-2, IL-4 and GM-CSF producing cells in progressive disease than in patients with nonprogressive disease and controls. A well preserved functional capacity to produce Th1 (IL-2, TNF-alpha, IFN- gamma and GM-CSF) and Th2 (IL-4) cytokines was noted. A relative increase of IL-2 and TNF-alpha producing T cells was observed upon unspecific in vitro activation in both non-progressive and progressive disease stages. CD4+ T cells from patients with progressive disease expressed in higher proportions CD54, surface(s) and cytoplasmic(c) CTLA-4 antigens as compared to patients with non-progressive disease and control donors. Lower frequencies of zeta chain and CD28 expressing T cells were found within the CD4 subset in progressive as well as non-progressive disease stages than in controls. The proportions of CD8+/zeta chain+ and CD4+/CD28+ T cells decreased by advancing stage. In opposite, increasing proportions of CD4+/CD54+, CD4+/sCTLA- 4+, CD4+/cCTLA-4+ as well as CD8+/sCTLA-4+ and CD8+/cCTLA-4+ T lymphocytes were noted by advancing stage.

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