|
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 and B cells through a variety of linked experimental
approaches. A variety of experimental approaches are taken to address the
theme issues. The approaches in current use examine 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 and function We
have shown previously that macrophages from X-linked immunodeficient (xid)
mice lacking functional Bruton’s tyrosine kinase (Btk) show poor induction
of macrophage effector functions such as NO induction, bactericidal activity
and secretion of proinflammatory cytokines (TNF-alpha, IL-1beta), but show
unchanged T cell priming functions such as induction of costimulatory molecule
expression (CD80, CD86) and enhanced induction of the T cell-directed cytokine
IL-12. Induction of some transcriptional regulators of the NF-kappaB family,
crucial for the expression of proinflammatory cytokines, is also poor in Btk-deficient
macrophages. Thus, Btk appears to be involved in signaling for inducible
effector functions, but not APC functions, in macrophages. Btk
is expressed in all cells of myeloid lineage, and in B cells, but not in T
cells. Therefore, we have examined the development and function of another
major cell of the myeloid lineage, the polymorphonuclear neutrophil
granulocyte (PMN), in Btk-deficient mice. We find that PMNs from xid mice show
deeply compromised functions. The induction of reactive oxygen species is
poor, as is the induction of NO. On the other hand the induction of
costimulatory molecule expression such as CD80 and CD86 is unchanged,
analogous to the findings on macrophages in these mice. Phagocytosis is
relatively little affected in these cells, as it is in macrophages. Even more
interesting is the finding that the numbers of PMNs in peripheral blood are
reduced by 2-3-fold in xid mice, and the PMNs observed are predominantly small
regular cells rather than the large irregular PMNs seen in wild-type mice. In
keeping with these findings, the maturation of PMNs in the bone marrow appears
to be hampered in xid mice, since irregular cells with high levels of the
granulocytic marker molecule Gr-1 are seen at lesser frequencies in them. Btk
is thus likely to be involved in granulocyte development as well as function. Role
of cAMP-mediated and reactive species (RS)-mediated pathways in controlling
death pathways relevant to T cell memory We
have shown previously that pentoxifylline (PF), a phosphodiesterase (PDE)
inhibitor in common clinical use, enhances long-term persistence of T cell
responses, including protective responses to a bacterial immunogen, Salmonella
typhimurium (Stm), via a cAMP-dependent protein kinase A (PKA)-mediated effect
on T cells if given to mice for a brief period during immunization, by
inhibiting post-activation T cell apoptosis. The pathways involved in the
death pathway which are affected by PF are thus of major importance. Data from
our group have shown that this death pathway is not mediated by death
receptors, but are mitochondrial, and are likely to be mediated by reactive
species (RS), - both oxygen intermediates and nitric oxide (NO). To examine if
the prevention of T cell death by PF operates via RS-mediated death, we have
now tested mice deficient in inducible nitric oxide synthase (iNOS). While the
secondary T cell responses generated in vitro by alloimmunisation are markedly
enhanced by PF for wild-type mouse T cells, PF has no effect on such a memory
response of iNOS-null T cells. These data indicated that PF probably affects
NO-mediated induction of post-activation T cell death. In keeping with this,
it would be expected that iNOS-null mice mount better memory T cell responses
than wild-type mice do, and our data with antigen-specific immunization in
vivo now demonstrate that, as late as six weeks after immunization, about
three-fold greater numbers of memory T cells, both CD4 and CD8, can be
identified in iNOS-null than in wild-type mice. Unnatural
peptide mimics as T cell immunogens The
retro-D-peptidic epitopes we have used for MHC class I binding and CD8 T cell
stimulation (designed by the structural biology group) have now been examined
in immunogenicity studies in vivo in terms of the bulk T cell repertoire
crossreactivity in immunised mice as well as the quantitative immunogenicity.
When mice are immunised with the native L-Oap [SIINFEKL; H-2-K-b-restricted],
CD8 T cell responses can be recalled efficiently in vitro with L-Oap itself,
as well as with retro-D-Oap, indicating that there is substantial
crossreactivity between Oap and its retro-D mimic even at the level of an
oligoclonal T cell repertoire triggered in vivo. However, contrary to
expectations, retro-D-Oap by itself does not appear to be a good immunogen in
preliminary experiments, since CD8 T cells from mice immunised with retro-D-Oap
do not yield efficient recall responses in vitro even with retro-D-Oap itself.
The basis for the poor immunogenicity is therefore of great potential
interest. Antigen
presentation pathway analyses We
have shown previously that proteins introduced into or synthesized in the
cytosol of antigen-presenting cells (APCs) such as macrophages can be
processed by proteasomal pathways and presented on MHC class II (MHCII) by
these cells. We have also shown a role for non-proteasomal proteolytic
cytosolic molecules such as tripeptidyl peptidases in this process. The
pathway by which peptides generated in the cytosol reach vesicular
compartments for loading onto MHCII has been thus an issue of interest. In
this connection, we have now observed that, while APCs from mice deficient in
the cytosol-to-ER transporter complex TAP do present cytosolically introduced
proteins well on MHCII, this presentation is not dependent on proteasomal
function, since pharmacological inhibition of proteasomes does not inhibit
this presentation to the degree observed in APCs from wild-type mice. This
finding suggests that there may be alternate pathways of protein processing
and peptide transport from the cytosol to vesicular compartments accessible
for MHCII loading, - one requiring proteasomes and TAP while the other may be
independent of these. We have also found that geldanamycin, an inhibitor of
HSP90, can substantially inhibit presentation of both exogenous and cytosolic
proteins on MHCII, implicating the HSP90 chaperone molecule in MHCII-mediated
antigen presentation. Publications Original
peer-reviewed articles 1.
John B, Rajagopal D, Pashine A, Rath S, George A and Bal V (2002) Role of
IL-12-independent and IL-12-dependent pathways in regulating generation of the
IFN-g component of T cell responses to Salmonella typhimurium. J Immunol
169:2545-2552. 2.
Suresh R, Vig M, Bhatia S, Goodspeed EPB, John B, Kandpal U, Srivastava S,
George A, Sen R, Bal V, Durdik JM and Rath S (2002) Pentoxifylline functions
as an adjuvant in vivo to enhance T cell immune responses by inhibiting
activation-induced death. J Immunol 169:4262-4272. 3.
Vig M, George A, Sen R, Durdik J, Rath S and Bal V (2002) Commitment of
activated T cells to secondary responsiveness is enhanced by signals mediated
by cAMP-dependent protein kinase A-I. Mol Pharmacol 62:1471-1481. 4.
Nair DT, Kaur KJ, Singh K, Mukherjee P, Rajagopal D, George A, Bal V, Rath S,
Rao KV, Salunke DM (2003) Mimicry of native peptide antigens by the
corresponding retro-inverso analogs is dependent on their intrinsic structure
and interaction propensities. J Immunol 170:1362-1373. |