The p]roduction and study of Theileria annulata macroschizont infected cells: relating to MHC class II expression, T cell stimulatory ability, cytokine mRNA production and their use as vaccines.

Theileria anmilata is a protozoan parasite of cattle of the genus Apicoinplexa, which causes the life threatening disease Tropical Theileriosis. In susceptible animals progression of this disease can be rapid with death occurring within 14 to 20 days post infection. T.annulata infection is characterised by the proliferation of macrophages (M0s) infected with the macroschizont stage of the parasite's life cycle. This stage is closely associated with pathology but may also be the main stage against which animals mount a protective responseThe aims of this thesis are to (1) examine the levels of MHC class II on infected cells, (2) attempt to relate MHC class II expression to their T cell stimulatory ability, (3) investigate the cytokine mRNA expression of infected cells and ascertain correlations between this and the T cell reactions they induced, (4) study the relationships between the cytokine mRNAs produced by infected cells and their potential effects on pathogenesis. In vitro, T.annulata infected cells possess augmented antigen presenting function and also the ability to activate resting naive/memory autologous T cells in a contact dependent manner. In vivo infected cells have been shown to congregate initially in the medulla of the draining lymphnode, where they associate with and activate T cells.This thesis investigates T cell stimulatory ability of T.annulata infected cells. Clonal populations of T.annulata infected cells were generated from CD14' monocytes and M9s, cultured and used to stimulate autologous naive T cells. T cell proliferation assays showed the clonal cultures possessed different T cell stimulatory abilities. Previous work showed that infected cells expressed elevated levels of MHC class II molecules. These molecules are involved in the stimulation of T cells and the possibility that the signals involved in nonspecific T cell activation were linked to MHC class II expression was investigated. Expression levels of MHC class II molecules by the clonal cultures did not correlate with the T cell stimulatory ability of the infected cells. After this finding the production of cytokine mRNA by infected cells was investigated. Cytokines are known to play major roles in the control of cellular activation/proliferation and immune responses.During this study the production of cytokine mRNA specific for: IL-la; IL-1P; IL-2; IL-4; IL-6; IL-10; TNFa and IFNy by clonal populations of infected cells were investigated using reverse transcriptase polymerase chain reaction (RT-PCR). Experiments showed that parasitised cells produce cytokine mRNAs typically associated with cells of the monocyte/M9 lineage. Limiting cycle RT-PCR was then employed to study differences in the levels of cytokine mRNAs produced by the different infected cell populations. This showed that variation in the T cell proliferation induced by the various populations of infected cells positively correlated with the levels of T cell stimulatory cytokine mRNAs produced by the infected cells. T cell proliferation assays showed the in vitro nonspecific T cell response peaks at day 5 post stimulation. The cytokine profiles of the responding T cells were then investigated from days 1 to 7. It was found that the T cell response following stimulation with infected cells always skewed to a Th, like response and that the differences in induced proliferation did not correlate with the levels of IL-2 and IL-4 produced by the responding populations. This suggested that infected cells possess the ability to manipulate T cell activity and that this would appear to occur via infected cell/T cell contact and T cell stimulatory cytokine production by the infected cells. Study also showed that infected cells initiate a none protective Thj like response, before leaving the node to enter the peripheryThe main method of control of T.annulata in many areas of the developing world is the immunisation of animals with attenuated T.annulata infected cell line vaccines. Long term in vitro culture of infected lines has been employed to produced successful vaccines. The mechanisms of attenuation are poorly understood. Following the findings that cytokine production by infected cells may have important consequences with respect to the induction of pathology and the control of immune responses during T.annulata infection, a clinical trial was performed to assess the efficiency of two of the clonal cultures as cell line vaccines. The two clones chosen (clones I and L) differed greatly in the levels of T cell stimulatory cytokine mRNA they produced and the levels of T cell proliferation they induced. Clone I induced low levels of T cell proliferation and produced low levels of cytokine mRNA, whilst clone L induced high levels of T cell proliferation and produced higher levels of cytokine mRNA. After inoculation of three animals with each of these cell lines and analysis of the data obtained (temperature, red/white blood cell counts, packed cell volume) it was found that the severity of the infection induced by clone L was greater than that induced by clone I. However, challenge of these animals some 60 days later showed all six to be equally resistant to infection with a lethal dose of T.annulata sporozoites.In summary, this thesis shows that the levels of T cell stimulatory cytokines produced by T.annulata infected cells correlate with the proliferative response of autologous T cells cultured with infected cells but that the levels of T cell proliferation do not correlate with the levels of autocrine cytokines produced by the proliferating T cells. The findings of this study also suggest that assessment of T cell stimulatory ability and cytokine mRNA production can aid in the selection of putative cell line vaccines