Abstract
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 response
The 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 periphery
The 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.
