|
Analysis
of antigen processing and presentation |
| Principal Investigator : Satyajit Rath
Project
Associates/Assistants Ph D Students
Collaborators The
aim of the ongoing linked projects in this group is to examine the generation
and activation of T, B and antigen-presenting cells using a number of
interlinked experimental systems. A variety of experimental approaches are
taken to address the theme issues. The approaches in current use examine
antigen-presenting cells (APCs) and pathways involved in antigen presentation
to MHC class I and class II-restricted T cells; and analyse the consequences
of intracellular signal transduction modulation for both development and
responses of B cells, T cells and macrophages using both genetic and
pharmacological tools. Role
of Bruton’s tyrosine kinase (Btk) in myeloid cell development We
have previously reported that the induction of many crucial transcription
factors is poor in macrophages from Btk-deficient XID mice. Proinflammatory
functional responses of macrophages are also compromised in the absence of
functional Btk, such as the induction of tumour necrosis factor-alpha (TNF-alpha),
inducible nitric oxide synthase (iNOS), or reactive oxygen intermediates (ROI).
Analogous to the data with macrophages, XID PMNs also showed deficient
induction of both ROI and NO. It has been previously shown that XID B cells
show enhanced apoptosis through a variety of pathways, and an analogous role
for Btk in macrophages was possible. Also, we have previously shown that a
lack of functional Btk leads to poor induction of transcription factors of the
NF-kB family, which are known to regulate apoptosis. Our data now show XID
macrophages die more rapidly in response to bacterial and cytokine ligands. Together,
these data indicate that the presence of a Btk-dependent signalling pathway is
required for some but not all functions of phagocytic cell lineages.
Phagocytic cells do not show optimal induction of microbicidal products such
as ROI or NO and die more easily in the absence of Btk, and this is likely to
lead to significant compromised innate immune responses responsible for
clearing microbial infections. Since
an absence of functional Btk thus leads to substantial reduction in
practically all proinflammatory functions of macrophages and PMNs, coupled to
an increased tendency to apoptosis, we examined the consequences on
inflammation in vivo. In a model of autoimmune inflammation in vivo
dependent on autoimmune Th1 responses, namely, experimental autoimmune
encephalomyelitis (EAE), XID mice showed poor induction of disease despite
their Th1 bias. In models of inflammation in vivo that do not depend on
adaptive immune responses, such as dextran sulphate sodium (DSS)-induced
colitis and carrageenan-induced footpad edema, XID mice are less susceptible
than WT mice are. The
absence of Btk also has developmental consequences for the myeloid lineage.
XID marrow shows a reduction in the frequency of granulocytic-myeloid
progenitor cells capable of colony formation in vitro, indicating that
Btk may be involved in the commitment to or expansion of the
granulocyte-monocyte progenitor cell population. Monocytic lineage cells are
fewer in the XID bone marrow than in wild-type (WT) marrow. While there is
little reduction at the earliest CD11blo
stage of granulocytic differentiation, there is progressive reduction in each
succeeding stage of granulocytic maturation as identified by progressive
acquisition of CD11b and loss of CD54 expression, indicating that Btk is
liable to play significant roles at multiple stages of granulocytic
differentiation. While human patients suffering from XLA do not necessarily
show an obvious neutropenia under normal conditions, infections frequently
induce neutropenic states in them, and this may be a consequence of either
enhanced apoptosis of myeloid cells, and/or reduced capability of the bone
marrow in responding to the increased demand for PMNs. Thus, Btk is likely to
be playing a significant, although partially redundant role at multiple points
during the development and functioning of the myeloid lineages, affecting the
outcome of many infectious as well as non-infectious inflammatory events in
vivo. This pleiotropy of functional roles raises the possibility that
subtle mutations in Btk may contribute significantly to the susceptibility of
carriers to both infectious and autoimmune diseases. Role
of lipid rafts in activation and differentiation of T cells In
order to dissect the roles of various signaling intermediates downstream of
the T cell receptor (TCR) in determining the various components of the
long-range cellular responses to activation, we have begun using MHCI-restricted
TCR-transgenic T cells (P14 TCR specific for the gp33-41 peptide of LCMV on
H-2Db),
along with target peptide-pulsed MHC-matched PS-SW epithelial line cells
growing adherently in vitro. The system allows us to terminate
antigenic exposure of T cells at various time intervals, and to examine the
continuing responses of the T cells both in culture and upon in vivo
transfer. Using this system, we have determined that as little as 3h of
exposure is sufficient for CD8 T cells to be activated, to express activation
markers, to proliferate extensively, and to become capable of cytotoxic
activity over the succeeding 96h or so in culture without any continuing
presence of stimulus. This
3h stimulation period allows us to examine the consequences of interruption of
various components of the signaling process. We have begun by examining the
role of membrane cholesterol, which is a crucial component of the so-called
‘lipid rafts’ thought to be important in a variety of signaling events
including those via TCRs. We have used methyl-beta-cyclodextrin (MCD), which
depletes membrane cholesterol, for this purpose. The concentrations of MCD
used, 3 mM and 6 mM, were determined to be capable of substantial inhibition
of very early events occurring within seconds and minutes of T cell
activation, such as the induction of calcium flux, increase in tyrosine
phosphorylation of proteins, and the formation of stable T cell-APC
conjugates. Activation
markers induced on the T cell surface by activation emerge over time; - CD69
is expressed early and disappears over subsequent hours, while CD25 arises
later and persists for longer. MCD treatment during the three hours of target
exposure prevents CD69 and CD25 induction at the normal times of induction.
However, these molecules are induced at later time points, so that by 48h
post-priming, the activation phenotype of MCD-treated cells catches up with
that of untreated cells. The T cells were also labeled with CFSE to measure
cell proliferation. Surprisingly, at high peptide concentrations (2 µg/ml),
MCD had practically no effect on the profile of cell proliferation, although
at lower peptide concentrations (200 ng/ml), it caused substantial inhibition.
Interestingly, while MCD-treated cells showed equivalent proliferation, the
absolute cell yields were much lower, suggesting that only some stimulated
cells underwent complete activation to proliferation under MCD cover, and/or
that cells triggered in the presence of MCD underwent death. However, when
assays for secondary effector functions were carried out on these triggered
cells, MCD treatment had little effect on either their ability to make IFN-gamma
in response to stimulation in vitro, or their ability to kill
peptide-labeled target cells in vivo. These data suggest that, firstly,
TCR-mediated cellular signaling is an actively continuing self-perpetuating
process for some time once initiated; secondly, that membrane cholesterol is
likely to play a role in early enhancement of the kinetics of this process
rather than being required for its initiation per se; and thirdly, that the
kinetic parameters of these signaling processes may be differentially linked
to distinct outcomes of T cell activation such as death, proliferation and
effector cell generation. Antigen
presentation pathway analyses We
now have evidence that a novel peptide transport mechanism is likely to be
involved in the translocation of peptides derived from cytosolic and
endogenous trans-membrane anchored proteins. We have shown earlier that the
MHCII-mediated presentation of proteins introduced into the cytosol by osmotic
lysis of pinosomes depends on cytosolic proteolytic mechanisms such as the
proteasome, and we have now examined the route and localisation of peptide
transport and loading onto MHCII molecules. We have shown earlier that
cytosolic protease activity is required for the presentation of endogenous
proteins, both cytosolic and trans-membrane anchored ones on MHCII. It is
hence possible that loading occurs in the ER lumen after peptide transport
from the cytosol using mechanisms related to those used by MHCI. We find that
peptide-MHCII complexes from endogenous proteins are not formed in the ER or
the Golgi complex. Peptide-MHCII complexes from proteins delivered into the
cytosol and those derived from trans-membrane anchored proteins are loaded on
MHCII in a large peri-nuclear compartment of the APC. This compartment
contains markers of lysosomes, LAMP-1 and cathepsin-D. Peptide-MHCII complexes
also do not localize with a marker of the late endosomes (MPR), further
defining the compartment where peptide loading occurs on MHCII in these cells.
There is almost immediate immunolocalization of the peptide-MHCII complex in
the lysosome after short cytosolic pulses, showing that while the peptides
from endogenous proteins are generated in the cytosol of APCs, they are
rapidly and possibly directly transported into lysosomal compartments followed
by loading onto MHCII. We have also begun examining the effects of the pleiotropic viral protein Nef from HIV-1 on APC functions. We find that, in addition to downmodulating MHCI and MHCII proteins from the APC surface as shown earlier by other workers, the costimulatory molecules CD80 and CD86 are also similarly downmodulated by Nef. The mechanisms and significance of these findings are now being actively pursued.
Publications Original peer-reviewed articles 1.
Raman VS, Akondy RS, Rath S, Bal V and George A (2003) Ligation of CD27 on B
cells in vivo during primary immunization enhances
commitment to memory B cell responses. J Immunol 171:5876-5881. |