INFECTION AND IMMUNITY cquired immunity by benbenzhou


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									                                    INFECTION AND IMMUNITY


1)      Formatting instructions/email application procedure:

        (a) Email applications should have “PhD Studentship Application” in the subject field, and
            should comprise:
            •   a covering letter
            •   CV
            •   a summary/abstract of any research project already undertaken (which should be no
                more than one side of A4)
            •   the names and addresses of two referees.
        (b) Save your application as a single Word document attachment. Name your Word
            document using your surname first and then your first name, eg Smith John.doc
        (c) Please indicate in your covering letter where you saw the advertisement and also where
            else you have looked for studentships.
        (d) Please state your nationality and how you will fund international fees if applicable.
        (e) Applications should be sent direct to the Research Degrees Administration Office

        Overseas applicants should also see FAQ1 below BEFORE submitting an application.

        Please note that if you apply without following the guidelines given above, your application
        may not be considered.

2)      Frequently Asked Questions

Q1.     Can students from outside the UK apply?

A.       Yes, overseas students have previously been accepted into the programme. The Child Health
Research Appeal Trust (CHRAT) will fully fund UK/EU students. Non-UK/EU students receive the normal
stipend, and the UK/EU component of their fees is paid, but they must pay the extra overseas fees
themselves (the current difference for 2008/09 is £12,730 per year and there is normally a 4 percent
increase on fees each year). Furthermore, all candidates who are selected for the Programme must be
interviewed, and we unfortunately have no money to pay for overseas students to come to interview.

Q2.     Can I come to visit the Institute before the interviews?

A.      Yes, there is an Open Day at ICH on Wednesday 19 November 2008 from 2.00pm onwards when
prospective applicants will meet the Postgraduate Tutors, existing PhD students, and have the opportunity to
take up tours of the facilities.

Q3.   I have a lower second degree but I am now doing an MSc. Is this equivalent to an upper

A.    Assuming you passed your MSc, you could be eligible to hold a PhD studentship. Please note,
however, that in the previous year’s applications nobody with such a background was successful.

Q4.    Does my age matter?

A.     No.

Q5.    I will be away at the time of the interviews (Monday 26th and Tuesday 27th January 2009).

A.     Contact us: we may be able to interview early.

Q6.    Do I need to have chosen my project from the portfolio, prior to interview?

A.     No. The best candidates will be selected and offered studentships and we will then arrange for you
       to come back to ICH and visit potential supervisors/labs before you make a final decision. However,
       if only one or two projects would be of interest to you because of your specialist background, please
       say so in your covering letter.

Q7.    I have another PhD offer, which needs a decision before you decide on your studentships.

A.     Ask them to wait (they usually will); if not, contact us.

                                    INFECTION AND IMMUNITY
3)     Project Descriptions:

 Retroviral vectors for gene therapy of lysosomal storage diseases (Dr Steven Howe)   4

 Lentiviral medicated zinc finger nuclease correction of immunodeficiency             5
 (Professor Adrian Thrasher)

 Hedgehog signalling in T-cell activation (Professor Tessa Crompton)                  6

 Functional consequences of gamma-delta T-cell phagocytosis (Dr Kenth Gustafsson)     7

Retroviral vectors for gene therapy of lysosomal storage diseases

Supervisors:    Dr Steven Howe

Hypothesis: Gene therapy has already proved to be an effective treatment for some forms of genetic disease,
including primary immunodeficiency. The clinical trials so far have used retroviruses as “vectors” to introduce genes
into the target cell population1. Because of some issues concerning the safety of these vectors we are now
investigating alternative gene delivery vectors. We now wish to extend the types of viruses which can be used for
gene delivery to include spumaviruses. The application of spumaviruses could be advantageous because they are
not associated with any know human disease or malignancy, and integrate their genetic payload into target cells in
a different pattern compared to other retroviruses which makes them potentially safer to use. The genome of these
viruses is probably more stable in quiescent cells (such as stem cells commonly used by our group) and the genetic
payload capacity is larger than existing retroviral vectors. Spumaviral vector technology has now advanced to the
point where disease models in large animals can be treated2.
     We intend to modify spumaviruses to enable their use in treating patients with genetic diseases. Our initial
target disorders will be lysosomal storage diseases, in particular Gaucher Disease3. This very severe disease is
often fatal and causes enlarged liver and spleen, skeletal and joint malformation, anaemia, neurological disorders
and occasionally reduction of immune function. Current treatments involve bi-weekly injections of the missing
enzyme, which is not effective or suitable for all patients. We hope that by modifying spumaviral vectors for gene
delivery will be able develop an improved treatment for Gaucher disease by gene therapy.

Aims and methods:         To optimise gene expression from spumaviral vectors we will clone in additional elements
such as promoters, regulatory elements and polyadenylation elements and test expression levels from the new
viruses with a reporter gene against existing vectors in vitro.
    In order to increase viral titres we will optimise production of spumaviral vectors. Currently ultracentrifugation is
used to concentrate virus, but alternative methods could be applied, including density gradients, HPLC and column
exchange. By attempting to alter the viral envelope it might be possible to adapt the efficiency of cellular targeting
and viral stability. We have experience of applying different envelopes to lentiviruses and gammaretroviruses.
    Once the vector system has been optimised we will clone in the glucocerebrosidase gene (the gene deficient in
Gaucher Disease) and test constructs using Western blots and real-time PCR to determine expression levels
    Once suitable vectors have been produced, they can be tested on Gaucher disease (glucocerebrosidase-
deficient) cell lines and assaying for recovery of enzyme levels. Cell lines have been developed from knockout
mice and patient’s cells that would be used to examine correction through available biochemical assays.
Lastly, techniques for delivery of virus or gene corrected cells would then be researched. Options include ex vivo
application of cells in artificial bio-scaffolds or transduction of blood / mesenchymal stem cells, or in vivo routes,
such as vector delivery to liver or muscle cells to act as an enzyme factory or to neurons to treat the neurological
aspects of the disease.

1. Gaspar,H.B. et al. Gene therapy of X-linked severe combined immunodeficiency by use of a pseudotyped
   gammaretroviral vector. Lancet 364, 2181-2187 (2004).
2. Bauer,T.R., Jr. et al. Successful treatment of canine leukocyte adhesion deficiency by foamy virus vectors. Nat.
   Med. 14, 93-97 (2008).
3. Guggenbuhl,P., Grosbois,B., & Chales,G. Gaucher disease. Joint Bone Spine 75, 116-124 (2008).

Lentiviral mediated zinc finger nuclease correction of immunodeficiency

Supervisors:    Professor Adrian Thrasher and Dr Waseem Qasim

Hypothesis:     Correction of inherited immunodeficiencies is possible by gene editing.

Introduction: Severe combined immunodeficiencies (SCID) usually present in the first few months of life and are
invariably fatal if untreated. Haematopoietic stem cell transplantation from a suitable HLA-matched donor can cure
SCID, though in the HLA-mismatched setting mortality and morbidity are high. Importantly, autologous bone marrow
stem cells can be harvested and gene modified ex-vivo, and then re-infused to mediate highly effective immune
reconstitution in vivo. The absence of pre-existing immunity abrogates the risk of immune mediated rejection and a
relatively small number of gene corrected stem cells can repopulate the entire T cell compartment, as the cells
acquire a notable survival advantage.
     To date clinical gene therapy has relied on a gene addition strategy, whereby a transgene (the interleukin
receptor common gamma chain (γc) in the case of X-linked SCID) integrates into the genome of haematopoietic
stem cells and is expressed downstream of a strong retroviral promoter element. In two clinical trails, unexpected
adverse events have recently been reported due to insertional mutagenesis, with five of twenty children with X-
linked SCID developing leukaemic T cell expansions. The genotoxic consequences of oncogene transactivation
following vector integration were rapidly characterised and the issue is currently being addressed through revision
of vector design with the incorporation of safer promoter elements and insulator sequences. However, concerns
remain that transformational changes may occurs, in particular in the context of the unregulated expression of
growth factor receptors such as γc.

Aims: Alternative strategies designed to correct mutations by gene editing, rather than the introduction of an
additional transgene, could eliminate the need for integrating vectors and greatly improve the safety profile of stem
cell gene therapy. One elegant approach which has recently been described has used zinc finger nucleases to
target a common mutation of the human γc gene. In the presence of appropriate homologous donor DNA
sequences, this can result in effective gene repair through up-regulation of homologous recombination (HR). To
date the strategy has required multiple plasmids or viral vectors for the intracellular delivery of two defined ZFN
(targeting opposite DNA strands), and the provision of a suitable donor template for HR. In an important recent
study, separate integration deficient lentiviral vectors were used to deliver the requisite elements, and showed
efficient gene correction of γc in cells lines and in primary cells.

Methods: Here we propose developing this approach further, through the generation of novel integration defective
vectors for the delivery of ZFN. This will be compared with refined gene addition strategies and use of less
mutagenic regulatory sequences. In vitro and in vivo model systems will be employed to test for toxicity and efficacy
of cellular reconstitution. This project will contribute to a wider pan-European collaboration investigating new gene
therapy technologies under a European Union Framework 7 award, and there will be access to important reagents,
cell lines and animal models through this collaboration. Current awards from the MRC, Wellcome Trust, Leukaemia
Research Fund and Department of Health are supporting related projects in the department.

1. H.B. Gaspar et al, Lancet 364, 2181-2187 (2004)
2. H.B. Gaspar et al, Mol.Ther. 14, 505-513 (2006)
3. R.J. Yanez-Munoz-et al, Nat.Med. 12, 348-353 (2006).
4. S.I. Thornhill et al, Mol.Ther. 16, 590-598 (2008)

Hedgehog signalling in T cell activation

Supervisors:    Professor Tessa Crompton and Dr Lucy Wedderburn

Hypothesis:     This project will test the hypothesis that Hedgehog (Hh) signalling modulates peripheral T cell

Aims and methods:         This project aims to test the hypothesis that Hedgehog (Hh) signalling modulates peripheral
T cell function. We aim to understand how Hh signalling influences T cell activation and to identify genetic targets
of Hh signalling in mature T cells. The Hh family of secreted intercellular signalling molecules specify cell fate and
patterning during embryonic development, and also regulate the homeostasis and renewal of adult tissues,
including skin, gut, lung and bone(1). Inappropriate activation of the Hh signalling pathway is involved in the
aetiology of many human cancers(2).
     We have recently shown that activation or inhibition of the Hh signalling pathway in T lymphocytes can influence
the outcome of T cell receptor (TCR) ligation, thereby influencing T cell activation and differentiation(3-6). Thus,
secretion of Hh proteins at sites of immune challenge or tissue damage may function to modulate the lymphocyte
response, preventing autoimmunty, and providing a micorenvironmental influence on T cell fate.
We hypothesise that in T cells, Hh signalling regulates a distinct set of genes which may be involved in influencing
the strength of the TCR signal. This project will investigate the role of Hh signalling in T cell activation by employing
our existing transgenic mouse models(3, 4). Functional studies will be performed to characterize the phenotype of
mature T cells from mice where Hh signalling is either a) active or b) repressed. Important functions including ability
to activate and proliferate, release of cytokines in response to activating signals, extent of cell death in response to
over-stimulation and ‘death’ signals, and the T cell effector phenotype will be investigated. In parallel, genetic
studies will be performed to investigate the expression of genes across the whole genome of resting or activated T
cells where Hh signalling is either active or repressed. Together it is anticipated that this study will characterize the
role of Hh signalling in T cell function and will reveal novel targets of Hh signalling in T cells, some of which may be
important in enhancing our understanding of how Hh signalling can lead to cancers.
 Methods employed in the project will include: Flow cytometry, Quantitative PCR, tissue culture, standard molecular
biology and biochemistry techniques, microarray analysis, bioinformatics.

1. Ingham, P.W., and M. Placzek. 2006. Orchestrating ontogenesis: variations on a theme by sonic hedgehog. Nat
   Rev Genet 7:841-850.
2. Taipale, J., and P.A. Beachy. 2001. The Hedgehog and Wnt signalling pathways in cancer. Nature 411:349-
3. Rowbotham, N.J., A.L. Hager-Theodorides, M. Cebecauer, D.K. Shah, E. Drakopoulou, J. Dyson, S.V. Outram,
   and T. Crompton. 2007. Activation of the Hedgehog signaling pathway in T lineage cells inhibits TCR repertoire
   selection in the thymus and peripheral T cell activation. Blood
4. Rowbotham, N.J., A.L. Furmanski, A.L. Hager-Theodorides, S.E. Ross, E. Drakopoulou, C. Koufaris, S.V.
   Outram, and T. Crompton. 2008. Repression of hedgehog signal transduction in T-lineage cells increases TCR-
   induced activation and proliferation. Cell cycle 7:904-908.
5. Rowbotham, N.J., A.L. Hager-Theodorides, A.L. Furmanski, and T. Crompton. 2007. A novel role for Hedgehog
   in T-cell receptor signaling: implications for development and immunity. Cell cycle 6:2138-2142.
6. Crompton, T., S.V. Outram, and A.L. Hager-Theodorides. 2007. Sonic hedgehog signalling in T-cell
   development and activation. Nature reviews Immunology 7:726-735

Functional consequences of gamma-delta T-cell phagocytosis

Supervisors:     Dr Kenth Gustafsson and Dr Siobhan Burns

Hypothesis: This project will test the hypothesis that gamma-delta T-cells (g/d T-cells) can phagocytose large
particles such as bacteria and synthetic beads and subsequently present associated antigens to both cytotoxic T-
cells via MHCI (cross-presentation) and to T-helper cells via MHCII, and that this uptake also leads to changes in
g/d T-cell phenotype, including cytokine production and cross-talk with other cells.

Aims and methods:          Gamma/delta T-cells are crucial part-takers in effector immune responses to both virus and
bacteria, at mucosal surfaces as well as in lymphoid organs (1, 2). Recently, they were shown to surprisingly also
have the capacity to function as professional antigen presenting cells (APC), joining the exclusive group of cells,
such as dendritic cells (DC) that were the only ones thought capable of stimulating naïve T-cells (3). The
mechanism of antigen uptake, processing and presentation by g/d T-cells is however totally unknown. In
combination with the fact that they are easily propagated ex vivo in large numbers, it is widely believed that these
cells could become an ideal target for future immunotherapeutic and gene therapeutic strategies (4, 5).
     In this Ph D student project, the role of g/d T-cells in the uptake of bacteria and synthetic beads as well as in the
subsequent presentation of MHC I- and II-restricted antigens to alpha-beta T-cells will be examined. The student
will also delineate how different modes of antigen uptake and processing by the gamma-delta T-cells affect alpha-
beta (a/b) T-cell activation, as well as how this affects the cells ability to secrete different cytokines.
     T-cell-mediated immune responses are important to protect against both viral (6) and bacterial (7) infections. In
order to mediate the T-cell reactivity, pathogen antigen presentation is required by APC. In addition, in vaccinations,
the same antigen presentation pathways need to be accessed in order to achieve an efficient immune response. It
is not clear which pathways and APC are the most efficient for different pathogens and vaccinations, and more
detailed knowledge could help improve different vaccine formulations. We recently showed that opsonisation with
pre-immune natural antibodies leads to increased uptake of virus into APC (i.e. DC) and increased presentation of
viral antigens to T-cells via MHCI cross-presentation (8). We have also shown in preliminary experiments that virus
(i.e. influenza), live or inactivated, can be taken up into g/d T-cells and quickly broken down and virus antigen
presented by both MHCI (cross-presentation) and MHCII to a/b T-cells. In addition, in other preliminary experiments,
we have shown that some g/d T-cells surprisingly appear able to phagocytose whole bacteria (i.e. E. coli), that
subsequently are quickly broken down in internal vacuoles, potentially ready for bacterial antigen presentation to a/b
     In this project, we will build on these results by examining more closely what happens with bacteria as well as
large synthetic beads when they encounter g/d T-cells. First we will examine what happens to the phenotype of g/d
T-cells following uptake of the different organisms by a study of cell surface markers. This will partly form a
continuation of the phagocytosis observations by employing both E. coli, and 1 micron synthetic beads as
phagocytic material for the g/d T-cells in an analysis of which particular g/d T-cells, and under what conditions, can
phagocytose. In the main part of the project, the student will investigate the ability of the g/d T-cells to present
antigens following delivery to the cells via both MHCI and MHCII and using specific T-cell hybridomas. During this
work, we will also employ a range of specific antigen processing inhibitors in order to understand which pathways
are used in the antigen processing and presentation. The student will also examine responses to the antigen-
presentation by g/d T-cells by primary a/b T-cells. In addition, we will investigate what cytokines are produced by
the g/d T-cells in response to the uptake.
     It is important to elucidate further the basic biology of interactions between virus and bacteria and the immune
system in order to develop both new therapeutics to reduce the severity of infection and new immunization
strategies to reduce incidence and transmissibility of infections. The discovery that g/d T-cells can take part in
pathogen antigen presentation to classical a/b T-cells is therefore important to investigate in much more detail.

1. Carding, S. R. and P. J. Egan. 2002. Gammadelta T cells: functional plasticity and heterogeneity.
Nat.Rev.Immunol. 2:336-345.
2. Hayday, A. and R. Tigelaar. 2003. Immunoregulation in the tissues by gammadelta T cells. Nat.Rev.Immunol.
3. Brandes, M., K. Willimann, and B. Moser. 2005. Professional antigen-presentation function by human
gammadelta T Cells. Science 309:264-268.
4. Moser, B. and M. Brandes. 2006. Gammadelta T cells: an alternative type of professional APC. Trends Immunol.
5. Bonneville, M. and E. Scotet. 2006. Human Vgamma9Vdelta2 T cells: promising new leads for immunotherapy of
infections and tumors. Curr.Opin.Immunol. 18:539-546.
6. Selin, L. K. and R. M. Welsh. 2004. Plasticity of memory T-cell responses to viruses. Immunity 20:5-16.
7. Malley, R., K. Trzcinski, A. Srivastava, C. M. Thompson, P. W. Anderson and M. Lipsitch. 2005. CD4+ T cells
mediate antibody-independent acquired immunity to pneumococcal colonization.            Proc.Natl.Acad.Sci.U.S.A
8. Durrbach, A., E. Baple, A. F. Preece, B. Charpentier, and K. Gustafsson. 2007. Virus recognition by specific
natural antibodies and complement results in MHC I cross-presentation. Eur.J.Immunol. 37:1254-1265.

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