|
Analysis of antigen processing and presentation |
| Principal Investigator :
Satyajit Rath Project Associates Ph D Students Collaborators
The
aim of the ongoing linked projects in this laboratory 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. A.
Control of T cell memory commitment by autocrine free radicals We
have shown earlier that the cAMP-mediated pathway can activate specific
commitment to long-term persistence of responsiveness during priming of human
and mouse CD4 T cells in vitro and in vivo, probably by
protecting activated T cells from apoptotic death through activation of
protein kinase A (PKA)-I. However, blocking the cAMP-PKA pathway during normal
priming of T cells does not modify either the induction of apoptosis or the
efficiency of commitment to memory. Therefore, while the PKA-I pathway is a
pharmacological mediator of T cell memory commitment, it does not normally
operate in this context. In order to examine the apoptotic pathways which
determine the normal limit to the induction of T cell memory, we have begun an
investigation of the various potential mechanisms that can possibly mediate
the death signal in activated T cells with respect to their relevance in T
cell memory commitment. Neither
caspase inhibition nor Fas-FasL blockade could modify the efficiency of
commitment of responding T cells to secondary responsiveness against
alloantigenic target cells. The primary proliferative T cell responses were
unaffected by these agents, but the secondary T cell proliferative responses
were in fact enhanced, suggesting that Fas- and caspase-mediated death
pathways could limit the actual responses mounted by secondary T cells, but
may not play a major role in the determination of the memory T cell pool size
during priming. On the other hand, MnTBAP, a metalloporphyrin known to be a
mimic of SOD and peroxidase as well as a peroxynitrite inhibitor, could
efficiently enhance T cell priming in vitro. However, it is not clear
if secondary proliferative responses of T cells in vitro can be taken
to reflect immune memory. We therefore examined if MnTBAP could induce any
physiologically relevant modulation of immune T cell memory in vivo.
Antigen-recalled T cell proliferative responses were enhanced if immunisation
was done under cover of a five-day treatment with MnTBAP in vivo. MnTBAP
did not inhibit induction of primary T cell proliferation, unlike the cAMP
pathway. However, it was efficient in blocking the induction of T cell
activation-induced cell death (AICD) in vitro. Thus, MnTBAP treatment
early during immunisation generates longer lasting T cell memory in vivo
by inhibiting AICD-mediated T cell loss rather than inhibiting T cell
activation per se. It
was not clear if MnTBAP was acting via the APCs or directly on T cells. We
approached this issue by light fixation of the APCs used for priming
alloresponses in vitro. Despite the inability of the fixed APCs to
undergo any activation, the presence of MnTBAP led to enhanced T cell
commitment to secondary responsiveness, indicating that its memory-enhancing
effect was mediated directly on the T cells. Thus, it appears that autocrine
free radical-mediated AICD may be crucial in limiting the degree of secondary
responsiveness in antigen-responding primary T cell populations. B.
Regulation of peptide-MHC complex generation and loss Subunit
vaccines consisting of peptidic epitopes have many advantages in terms of
production and stability as well as precision of the triggered immune
responses. However, effective immunogenicity of subunit vaccines has
significant problems; - such small epitopic immunogens have short half-lives,
and they need adjuvants. Long-lived peptides with high affinities for MHC may
be able to ameliorate some of the difficulty. Further, increasing the
longevity of antigen-specific peptide-MHC complexes on APCs in vivo
increases their Th1 immunogenicity, which is useful for protection against
intracellular pathogens, which are major public health problems, such as
infections with Mycobacterium, Salmonella and Leishmania.
Increasing the peptide-MHC complex density can be achieved by increasing the
affinity of the peptide for the MHC molecule, and prolongation of the complex
availability can be achieved by making the epitopes less susceptible to
degradation. For both these possibilities, the structural basis of peptide
mimic design needs to be established and tested in such systems, - an approach
being used by our collaborators in structural biology. We have been using such
designed non-degradable peptide mimics as antigens. In
such biological assays for peptide binding to MHC class I (MHCI), the retro-D
forms of both an MHCI-binding peptide from ovalbumin (Oap) and an MHCI-binding
peptide from a VSV glycoprotein (VSVp) bound well to the MHCI molecule,
H-2K-b. The MHC-contacting residues of Oap that have been identified for the
native L-form of the peptide were next tested in the retro-D form, and the
residue dependence pattern was found to be similar, at least for two residues
tested so far, supporting the prediction made in the analytical approach that
the mode of interaction of retro-D peptides with MHC class I is similar to
that of the native L-forms. In
the biological assays for recognition of these peptide-MHC complexes by T
cells, the retro-D-Oap stimulated Oap-specific T cells very well. The
D-mutations in the retro-D-Oap also stimulated the T cell line to the same
extent that they could bind to H-2K-b, further supporting the similarities in
binding and recognition of the two forms. These
studies were extended to the intracellular mechanisms for peptide loading on
MHCI. L-Oap and retro-D-Oap were both loaded cytosolically by using the
technique of osmotic lysis of pinosomes. While the presentation of L-Oap was
dependent on the ABC-family peptide transporter TAP, the retro-D-Oap was well
presented cytosolically independent of TAP. This suggested that the rate
limiting factor for TAP-independent diffusional penetration of cytosolic
peptides into the ER for MHC class I loading was likely to be the proteolytic
degradation of the peptides in the cytosol. This was supported by the finding
that proteasomal inhibitors allowed L-Oap also to be presented well
cytosolically in a TAP-independent form. Finally,
the issue of the life-span of the peptide-MHC complexes on APCs was addressed
by examining the half-life of either L-Oap or retro-D-Oap on APCs, and
unequivocal data were obtained showing that retro-D-Oap survives for much
longer periods than L-Oap does, supporting the speculation that this approach
could modify the immunogenicity of peptidic antigens. Publications Original
peer-reviewed articles 1.
Mukherjee P, Dani A, Bhatia S, Singh N, Rudensky AY, George A, Bal V,
Mayor S and Rath S (2001) Efficient presentation of both cytosolic and
endogenous transmembrane protein antigens on MHC class II is dependent on
cytoplasmic proteolysis. J Immunol 167:2632-2641. 2.
Bhatia S, Mukhopadhyay S, Jarman E, Hall G, George A, Basu SK, Rath S,
Lamb JR and Bal V (2002) Scavenger receptor-specific allergen delivery elicits
IFN-g-dominated
immunity and directs established TH2-dominated
responses to a nonallergic phenotype. J Allergy Clin Immunol 109:321-328. Miscellaneous 1.
Rath S (2001) Opportunities for science careers in India: an invitation
to the Mad Hatter’s tea party. Economic & Political Weekly 36:1372. |