Regulation of T-cell proliferation and B-CLL apoptosis by CD6 and FAS/FASL

Sammanfattning: T-cell activation involves the interaction between the T-cell receptor (TCR) and the peptide/MHC complex presented on an antigen-presenting cell. However, this is not sufficient for the full development of T cell responses and co-stimulatory signals from accessory molecules are required. In the first part of this thesis, the role of CD6 as accessory molecule in T-cell activation and proliferation was investigated. In the first study, three different epitopes associated with the CD6 molecule were defined by using a panel of five CD6 mAbs, all of which were capable of enhancing TCR/CD3 complex-mediated T-cell proliferation. The new epitope, defined by IOR-T1 mAb, appeared to be non-conformational and consistently induced greater responsiveness in resting T cells stimulated by suboptimal CD3 antibodies. This study provides evidence for the existence of functionally distinct epitopes on the CD6 molecule with co-stimulatory capacity in T-cell proliferation. In the second and third studies we explored the intracellular signaling pathway in CD6-mediated T-cell co-stimulation, and we demonstrated that one of the earliest biochemical events involves protein tyrosine kinase (PTK) activation. In addition, the downstream signaling pathway appeared to be mediated by PKC and the Ca2+-dependent phosphatase calcineurin, which results in IL-2-dependent T-cell proliferation. In the third study, IL-2-dependent T-cell proliferation was shown to be induced by simultaneous ligation of the CD6 and CD28 accessory molecules via apparently distinct signaling pathways. Taken together, we provide evidence suggesting an important role for CD6 in the regulation of T-cell activation and proliferation. The CD6 molecule is also expressed on B-cell chronic lymphocytic leukemia (B-CLL) and a subset of normal B cells, however its function in B cells has not been determined. B-CLL is characterized by the progressive accumulation of monoclonal B cells, which may be the result of several factors leading to extended B-CLL cell life span, increased proliferative capacity and diminished cell death. The second part of this thesis addresses the function of CD6 and the involvement of the Fas/FasL system in the growth control and clinical progression of B-CLL cells. In the first of these studies, we found CD6 surface expression on most B-CLL cells, although the levels were unrelated to clinical progression. CD6 was not involved in B CLL proliferation in vitro, but rather in the protection of B-CLL cells against apoptosis induced by anti-IgM antibodies. CD6 cross-linking counteracted anti-IgM-induced Baxa mRNA and protein up-regulation, resulting in an increased Bcl-2/Bax ratio. We suggest that CD6 may provide survival signals to B-CLL cells through the modulation of the Bcl-2/Bax ratio. In a second study we found high levels of sFas in B-CLL sera, which were correlated with progressive disease and late clinical stages. It was also shown that B CLL cells constitutively express low levels of Fas and no FasL on the surface, while expressing mRNA for both. In vitro activated B-CLL cells expressed both Fas and FasL and released the soluble forms. However, Fas-positive B CLL cells were resistant to anti-Fas induced apoptosis. Finally, we found killing of Fas expressing Jurkat T cells by sFasL released from B-CLL cells. Taken together, it is suggested that the features of the Fas/FasL system in B CLL and the survival signal generated from CD6-CD6 ligand interaction, may contribute to the accumulation of leukemic cells and the progression of the disease, by down-regulating the apoptotic response.