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Home > Annual Report > 2004 | |||
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Introduction The
Lausanne branch has continued to focus its activities on the molecular
and cellular mechanisms that regulate immunity mediated by T cells, natural
killer (NK) cells and NKT cells. Given the complexities of the innate
and adaptive immune systems, a significant part of the laboratory research
program involves studies in mice that aim at the elucidation of the variety
of recognition strategies used by lymphoid cells. As a result of the recent
progress in the identification of tumor antigens recognized by CD8+ T
cells in cancer patients, a strong emphasis of the branch laboratory research
program deals with the basics of human cancer immunology. This program
is closely integrated with our growing clinical program, which is conducted
as a part of the Institute’s Cancer Vaccine Program.
Cell Fate Determination Group The Cell Fate Determination Group led by Dr. Freddy Radtke is interested in the molecular mechanisms controlling stem cell maintenance, lineage commitment and differentiation in self-renewing systems such as the hematopoietic system, the skin and the gut. In addition this group investigates the potential role of these mechanisms in tumorigenesis. Current attention is focused on the Notch signaling pathway, which regulates numerous binary cell fate decisions in diverse organisms. Previous studies by the group using conditional gene targeting strategy have established an essential role for Notch1 in specifying the T cell lineage. In addition a novel function for Notch1 has been uncovered at a later stage of T cell development, where it is involved in the control of VDJ rearrangement at the T cell receptor (TCR) beta locus. In the skin an unexpected function of Notch1 as a tumor suppressor gene has been discovered. In the intestinal tract Notch signaling is required for the maintenance of undifferentiated proliferative cells in crypts thereby acting as a gatekeeper of stem/progenitor cells.
Developmental Immunology Group The
Developmental Immunology Group led by Dr. Rob MacDonald continues to be
interested in the development of T cells and in particular a subset of
nonconventional T cells known as NKT cells. NKT cells have recently emerged
as an important regulatory T cell subset implicated in autoimmunity and
tumor immunity. In contrast to conventional T cells NKT cells utilize
a semi-invariant aßTCR to recognize glycolipids presented by the
monomorphic CD1d molecule. In order to study thymic selection of NKT cells
the group has produced transgenic mice expressing CD1d either on CD4+
CD8+ cortical thymocytes or on thymic dendritic cells. Positive selection
of NKT cells was found to be mediated exclusively by cortical thymocytes
whereas negative selection was mediated primarily (but not exclusively)
by dendritic cells. Thus, like conventional T cells, the NKT cell repertoire
is shaped by both positive and negative selection.
Innate Immunity Group The Innate Immunity Group led by Dr. Werner Held studies the development and function of NK cells. NK cells play important roles in innate immunity to infection and tumor cells. NK cell-mediated lysis of target cells is regulated by a dual receptor system, which integrates signals from activating receptors and inhibitory receptors. The latter interact with Major Histocompatibility Complex (MHC) class I molecules expressed on target cells. Surprisingly the group found that the inhibitory Ly49A NK cell receptor not only binds to the MHC class I ligand expressed on potential target cells (i.e. in trans) but is constitutively associated with MHC class I in cis (i.e. on the same cell). Cis association and trans interaction occur via the same binding site. Consequently, cis association limits the number of Ly49A receptors available for binding MHC class I on target cells, thereby dampening NK cell inhibition via Ly49A. By lowering the threshold at which NK cell activation exceeds NK cell inhibition, cis interaction seems to allow an optimal discrimination of normal and infected or transformed host cells.
Molecular Immunology Group The
main research interest of the Molecular Immunology Group led by Dr. Immanuel
Luescher is focused on the molecular mechanisms involved in CD8+ cytolytic
T lymphocyte (CTL) recognition of, and activation by, peptide-MHC (pMHC)
complexes. The current research effort is based on the preparation of
well defined soluble pMHC complexes and their testing on CD8+ T cells.
Initially the group prepared and studied dimeric pMHC complexes containing
linkers of different length connecting the a3 C-termini of the two pMHC
entities. Findings obtained include: (i) pMHC dimers containing short
linkers (10-30 Å) efficiently bind to and activate cloned CTL, whereas
those containing long linkers do not; (ii) pMHC dimers containing short,
but not those containing long linkers, induce rapid death of activated,
but not naïve, CD8+ T cells. This effect is not based on classical
death mechanisms (e.g. granzyme/perforin-, Fas-, TRAIL- or TNFRmediated),
but relies on cell activation-dependent mitochondrial dysfunction and
most likely involves Bcl2 pro-apoptotic family members (e.g. BIM and BNIP3);
(iii) pMHC complexes containing long rigid linkers not only fail to trigger
CD8+ T cells but effectively inhibit CTL-mediated cytotoxicity; (iv) based
on cell activation and binding studies, FRET experiments and computer
assisted docking experiments, the group discovered a binary binding mode
by which two TCR molecules engage in an anti-parallel manner to two pMHC
complexes facing each other with their constant domains. Subsequent studies
with well defined tetramers and octameric pMHC complexes confirmed these
findings. Notably, it was found that octameric pMHC complexes with sub-nanomolar
binding constants that contain short linkers induce CTL death, whereas
those containing long rigid linkers completely inhibit CTL-mediated cytotoxicity.
This work, done in collaboration with groups at the Swiss Federal Institute
of Technology Lausanne (EPFL), uses single molecule microscopy as a new
tool to investigate peptide presentation by antigen-presenting cells as
well as peptide recognition by CTL on a molecular level. Antigen Processing Group The
Antigen Processing Group led by Dr. Frédéric Lévy
is involved in the analysis of the intracellular events that contribute
to the production of CTLdefined tumor antigens. The group has shown previously
that the HLA-A*0201-restricted immunodominant peptide derived from Melan-A,
the target of vaccine trials in the branch, is inefficiently processed
in cells expressing the immunoproteasomes, a type of proteasome that is
constitutively expressed in dendritic cells and induced in many other
cells upon exposure to IFN-?. Immunization of HLA-A2 transgenic mice with
third generation recombinant lentiviral vectors expressing the minimal
antigenic determinant of Melan-A induced a potent T cell response, which
was detectable ex vivo. In contrast, only a modest response was detected
after immunization with lentivectors containing the sequence of the full
length Melan-A protein. To test whether the expression of immunoproteasomes
contributes to this poor response, the group performed similar experiments
in HLA-A2 transgenic mice in which the gene coding for LMP2, one of the
catalytic subunits of the immunoproteasome, has been deleted. These mice
were generated in collaboration with the group of Dr. B. Van den Eynde
(LICR, Brussels branch). Contrary to the response observed in HLA-A2 transgenic
mice, immunization of these HLA-A2 LMP2-/- mice with lentivectors containing
the full-length Melan-A sequence resulted in a T cell response that was
indistinguishable, both in amplitude and quality, from the response after
immunization with lentivectors containing the sequence of the minimal
determinant. These results demonstrate that the expression of immunoproteasomes
negatively affects the processing of Melan-A, thereby influencing the
anti-Melan-A T cell response in vivo. Molecular Tumor Immunology Group Research
activities of the Molecular Tumor Immunology group led by Dr. Jean-Charles
Cerottini are devoted to the characterization of human tumor antigens
that are recognized by CD8+ T cells from melanoma patients. One project
aims at identifying the function and regulation of the melanocyte lineage
specific protein Melan-A, a small transmembrane protein that is the target
of vaccine trials in the branch. Melan-A is present in the trans-Golgi
network and in early stage melanosomes of pigmented cells. The group has
generated Melan-A in vitro knock-down cells using lentivirus-delivered
siRNA as well as Melan-A overexpressing cells. Characterization of these
cells showed that Melan-A, in contrast to other melanocyte-specific proteins,
has a negative role in pigmentation. Expression of tyrosinase, the key
enzyme in melanin synthesis, was not altered in Melan-A knock-down or
overexpressing cells, suggesting that Melan-A may affect melanogenesis
by modifying the melanosomal environment. In addition, it has been found
that Melan-A is mono-ubiquitylated and interacts with two members of the
HECT-E3 ubiquitin ligase family. Blocking Melan-A ubiquitylation decreased
the half-life of the protein and affected its localization. Interestingly,
this also influenced melanin content in melanocytic cells. The results
suggest a role of ubiquitylation in the sorting of Melan-A from melanosomes
to multi-vesicular bodies. These findings also highlight the importance
of ubiquitylation processes in the physiology of pigmented cells.
Clinical Tumor Immunology Group The
Clinical Tumor Immunology Group led by Dr. Pedro Romero continues its
studies on both naturally acquired and vaccine induced CD8+ T cell responses
to well defined melanoma tumor antigens. To this end, the group is investigating
the relationship between the T cell surface phenotype and functional stage
of differentiation. While it is widely accepted that T cells not previously
exposed to antigen in the periphery co-express the high molecular weight
isoform of CD45, CD45RA, and the SLC/ELC chemokines receptor CCR7, the
group has identified a subset of cord blood CD8+ T cells that does not
express the latter molecule. Such subset is rare or does not exist at
all in peripheral T cells in adult individuals. Detailed characterization
of these cells conclusively showed that they were naïve T cells and
already present in the mature single CD8+ thymocyte population. We suggest
that these cells represent precursors of naïve T cells programmed
to enter peripheral tissues (e.g. the skin) that may play a role in the
acquisition of peripheral tolerance during the neonatal phase of life,
such as demonstrated in mouse models. This year the group has also concluded
a major study assessing the functional properties of antigen-experienced
CD8+ T cells in various tissues obtained from a relatively large group
of cancer patients. Taking advantage of the high frequencies of Melan-A-specific
T cells often occurring in HLA-A2 patients with advanced melanoma, the
group has extensively phenotyped these cells and isolated them directly
from freshly dissociated tumor masses for functional assays such as lytic
activity, cytokine secretion and expression of effector function associated
genes. It should be stressed that this study is unique in that it avoids
in vitro expansion of the antigen specific T cells, thus allowing to acquire
information on the functional status of T cells infiltrating human tumors.
The major finding was that T cells recovered from tumor masses are hyporesponsive.
Indeed, they have reduced levels of perforin and lytic activity and they
are unable of releasing IFN-? upon antigen challenge. However, they are
still able to produce IFN-? when the stimulus bypasses the T cell receptor.
Importantly, hyporesponsiveness is reversible upon short term in vitro
culture. The cells also display significant levels of proliferation when
cultured in the presence of cytokines. An additional interesting finding
was that the T cells with the same antigen specificity that were recovered
from the circulating compartment from the same patients appeared functionally
competent thus pointing to a tumor associated state of functional tolerance.
These findings may help explaining, at least in part, the paradox of tumor
progression in patients with apparently vigorous responses of circulating
tumor antigen-specific T cells. They raise two important issues. First,
the critical importance of monitoring T cell function, and not jut T cell
frequencies, in the tumor microenvironment. And, second, the need to identify
strategies to overcome blunting of the specific T cell response within
the tumor for vaccines to be clinically effective.
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