Multiple sclerosis (MS) is believed to be an autoimmune, inflammatory, demyelinating disease of the central nervous system (CNS), resulting in myelin degradation, loss of the myelin forming cell, the oligodendrocyte (ODC), and axonal degeneration. The hypothesis underlying this work is that gammadelta T cells play a distinct role in MS pathogenesis by initiating, perpetuating, or regulating the immune response directed against the myelin/ODC unit. Initial comparative experiments utilising short term gammadelta T cell lines from peripheral blood (PB) and cerebrospinal fluid (CSF) of MS and other neurological disease (OND) patients, indicated no significant gammadelta subtype or cytotoxicity differences between cells derived from PB and CSF compartments or between MS and OND patients. During the course of these studies, it became apparent that the variably short in vitro lifespans of PB and CSF gammadelta T cells represented a major limitation that hampered further comprehensive studies. Efforts to increase their culture lifespans through restimulation regimens were only marginally successful, so an alternate immortalisation strategy using Herpesvirus saimiri (HVS) was employed. This study reports for the first time, the successful HVS growth transformation of human gammadelta T cells derived from both MS PB and CSF. Multiple HVS transformed PB and CSF gammadelta T cell (t -gammadelta T cell) lines and clones were generated, and have existed in IL-2 dependent culture for periods in excess of 2.5 years without the need for additional stimulation. Comparative analysis of a series of t-gammadelta T cell lines and their personal non-transformed lines indicated the transformation process did not alter their surface molecule expression, cytokine, or cytotoxicity responses. Cell surface characterisation of the t-gammadelta cell lines and clones demonstrated HVS was capable of immortalising a wide spectrum of gammadelta TcR subtypes, along with identifying t -gammadelta T cell clones displaying rare gammadelta T cell phenotypes, not commonly studied. MS t-gammadelta T cells uniformly expressed proinflammatory cytokine profiles (IFN-gamma, TNF-alpha), but only minimal IL-2 or anti-inflammatory IL-4 and IL-10. All gammadelta TcR subtypes displayed identical cytokine pattern expression, suggesting that cytokine expression is independent of gammadelta T cell subtype. t-gammadelta T cell lines demonstrated a graded cytotoxicity towards a panel of tumour cell targets, ranging from high (U937, Jurkat) to moderate (KG-1) to poor (Colo-205). Similar killing patterns of tumour targets were observed for subtype specific t-gammadelta T cell clones, and antigen recognition studies indicated a graded recognition of tumour cell ligands, together suggesting that both cytolytic function and tumour cell recognition, as was seen with cytokine profiles, are independent of gammadelta T cell subtype. In contrast, only Vgamma9Vdelta2 positive t-gammadelta T cells responded to candidate non-peptidic phospholigand and alkylamine antigens. No MS t-gammadelta T cell reactivity was observed to putative MS antigens myelin basic protein, alphaB-crystallin, or Chlamydia pneumoniae. (Abstract shortened by UMI.)
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/9045 |
| Date | January 2001 |
| Creators | Pon, Robert A. |
| Contributors | Freedman, Mark, |
| Publisher | University of Ottawa (Canada) |
| Source Sets | Université d’Ottawa |
| Detected Language | English |
| Type | Thesis |
| Format | 192 p. |
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