de Guzman, Sarah Yvonne
Description
Membrane transfer is the cell-to-cell contact dependent exchange
of plasma membrane and surface molecules between cells. It has
been described for a wide range of immune cells, nevertheless the
molecular mechanisms mediating this exchange remain unclear. The
studies outlined in this Thesis provide new insights and a better
understanding of the transfer process.
Previous studies in my supervisor’s laboratory showed that
activated antigen- specific B cells and T...[Show more] cells readily donate
their antigen-specific receptors to neighbouring lymphocytes of
an unrelated specificity via plasma membrane exchange. Results
obtained in this Thesis confirmed and further characterised
several properties of antigen receptor transfer as well as
suggesting new potential mediators of membrane exchange.
The results in Chapter 3 detail important properties of membrane
exchange between B cells, and based on these unique features
propose a molecular mechanism for this process. It was confirmed
that B cells require appropriate activation conditions in order
to donate membranes and cell surface proteins, with naïve B
cells being much less capable of transfer. Additionally, it was
confirmed that activated B cells are capable of transferring
membranes and surface proteins at 4oC, thus excluding many of the
currently proposed energy dependent mechanisms. Chapter 3
describes the investigation of an energy independent mechanism of
exchange involving cell penetrating peptide (CPP) motifs. The
results obtained demonstrate that analogous to transfer between B
cells, CPPs are up to 40-fold more efficiently taken up by
activated, rather than naïve, B cells and that this uptake
occurs at both 37oC and 4oC. These data suggest that receptors
involved in the initiation of membrane transfer may contain CPP
motifs important in facilitating this process.
Chapter 4 describes attempts to identify the molecular basis of
membrane transfer via a comparative gene expression analysis.
This study took advantage of the finding that B cells require
appropriate activation conditions to achieve efficient levels of
membrane transfer. In an effort to unravel the proteins involved
in this process, transcriptomes of LPS stimulated B cells, which
are known to efficiently transfer membranes, were compared with
the transcriptomes of CpG stimulated and unstimulated B cells
which are less efficient at transferring membranes.
Bioinformatics analysis identified five proteins containing CPP
motifs that may play a role in B cell membrane transfer, namely
ALCAM, AMIGO2, CTLA-4, Slp3 and TIGIT. Monoclonal antibodies
(mAbs) specific for ALCAM, CTLA-4, TIGIT and the TIGIT ligand,
PVR, demonstrated increased expression of all these proteins on
the cell surface of LPS activated B cells. Furthermore,
incubation of B cells with ALCAM, TIGIT and TIGIT + PVR mAbs
resulted in significantly enhanced membrane transfer, whereas the
CTLA-4 mAb, known to block CTLA-4 binding, had no effect. Based
on these data and other published findings it is hypothesised
that mAbs specific for ALCAM and TIGIT/PVR may crosslink these
receptors, creating a patching effect, similar to a lipid raft,
in which there are areas of localised membrane destabilisation
and, consequently, more accessible areas for CPP-mediated
membrane fusion.
Based on the current understanding of membrane transfer it should
be possible to harness this phenomenon to enhance antigen
specific CTL responses in the adoptive immunotherapy of
established tumours. Thus, experiments described in Chapter 5
used an adoptive T cell immunotherapy model to investigate
whether TCR sharing can be harnessed to control tumour growth.
The model utilised the ovalbumin (OVA)-expressing EG7 (EG7-OVA)
thymoma cell line to establish tumours in mice. These tumours are
susceptible to killing by OVA-specific TCR transgenic (OT-I) CTL,
thus resulting in a reduction in EG7-OVA tumour volume. To
establish the influence of antigen receptor transfer in the
control of tumour growth, perforin deficient OT-I CTLs, which
have been shown previously to be less effective at bringing about
tumour rejection due to their lack of perforin, and CTLs of an
unrelated specificity (B6.SJL.TCRP14 CTLs) were used. Transferred
alone each of these CTL populations should be incapable of
inducing tumour regression. However, when adoptively transferred
together if TCR transfer occurs the B6.SJL.TCRP14 CTL should
acquire OVA-specific TCR from the perforin deficient OT-I CTLs
and gain the capacity to recognise and eliminate the EG7- OVA
tumour cells and thereby mediate tumour regression. The results
obtained, however, demonstrated that perforin deficient OT-I
CTLs, but not B6.SJL.TCRP14 CTLs, were as efficient as wild type
OT-I CTLs at eliminating established EG7-OVA tumours in vivo.
This finding indicated that the CTLs used in this model do not
require perforin to control tumour growth, with subsequent in
vitro studies suggesting involvement of the Fas/FasL pathway.
Furthermore, results described in this Chapter demonstrate that
extrodinarily small numbers of CTLs within tumours can induce
tumour regression, suggesting that in vivo many other mechanisms
work in concert to control EG7-OVA tumour growth, NK cells being
the most obvious. In addition, variations in the results obtained
with the EG7-OVA cell line in different laboratories may be due
to genetic drift of the cell line, thus highlighting the
importance of stringent cell line authentication.
In conclusion, this Thesis identified several candidate proteins
required for efficient membrane transfer between B cells as well
as providing evidence for a potential membrane exchange mechanism
involving proteins that contain CPP motifs that facilitate
membrane fusion and thus the transfer of membranes and associated
cell surface proteins.
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