|
Analysis of antigen processing and presentation |
|
Principal Investigator : Satyajit Rath Project Associates Ph D Students Collaborators The aim of the ongoing activities in this laboratory
is to examine the generation and activation of T and B cells through a variety
of linked experimental approaches. Some of the approaches in current use are;
delivering protein antigens to antigen-presenting cells (APCs) via scavenger
receptor-like ligand specificities to examine the effects of such delivery on
antigen processing, immunogenecity and tolerance; examining APCs and pathways
involved in non-classical antigen presentation to MHC class I and class
II-restricted T cells; analyzing 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; and
characterizing the receptor-ligand interactions of some novel non-canonical
quasi-T cell lines. Macrophage effector functions controlled by Bruton’s tyrosine kinase (Btk) Our previous studies have indicated that LPS-mediated induction of IL-12 is better in xid than in wild-type macrophages as an indirect consequence of poor iNOS induction in Btk-deficient xid macrophages. Since antigen presentation and costimulation was comparable between xid and wild-type macrophages, Btk appeared to play little role in the induction of T cell priming functions in macrophages. On the other hand, the bactericidal ability of xid macrophages was much poorer than that of wild-type cells. Thus, in 2 h of incubation, the wild-type cells reduced bacterial viability by over 98%, while more than one-third of the input bacteria were still alive in the xid macrophage cultures (p<0.01). The poor bactericidal activity in xid macrophages is also accompanied by a deficit in bacterial phagocytosis. The induction of reactive intermediates such as NO or reactive oxygen intermediate species is also defective in xid macrophages. Upon stimulation, macrophages secrete TNF-a and IL-1b,
two major pro-inflammatory cytokines. The levels of induction of both
cytokines (TNF-a and IL-1b) were higher in wild type than in xid macrophages,
while higher levels of IL-12 were induced from xid macrophages. Increased
induction of IL-12 from xid macrophages is a consequence of the poor induction
of iNOS and NO. However, inhibition of iNOS in LPS-activated macrophage
cultures did not change the levels of TNF-a or IL-1b induction in either wild
type or xid macrophages, suggesting that the effect of Btk on TNF-a and IL-1b
induction was NO-independent. Since the NF-kB family of transcriptional
regulators have been shown to be important for microbicidal activity as well
as the induction of expression of both TNF-a and IL-1b, we examined if xid
macrophages also showed deficient induction of nuclear translocation of NF-kB.
Induction of all three members of the rel family by LPS could be clearly
observed in the case of wild-type but not in xid macrophages. We have shown previously that anti-microfilarial (mf)
T cell responses in xid mice are skewed towards the production of Th1
cytokines such as IFN-g as a result of enhanced IL-12 secretion. Since xid
mice clear injected mf far more slowly than wild-type mice, it was possible
that Th1 T cell responses led to delayed-clearance of introduced mf in xid
mice. In order to examine this possibility further, we purified T cells from
mf-infected wild-type or xid mice and transferred them into naive wild-type or
xid mice, which were then given mf to estimate the kinetics of mf clearance.
Wild-type mice showed unaltered rates of mf clearance even if they had been
given T cells from mf-infected xid or wild-type donors, and xid recipients
showed delayed clearance despite receiving T cells from wild-type mf-infected
donors. Thus, the T cell cytokine profile was not of crucial importance in
causing the difference between mf clearance rates of wild type versus xid
mice. Similarly, clearance of microfilaraemia was unchanged between wild type
and IFN-g-/- mice. These data reinforce the relative lack of significance of
particular T cell cytokines in controlling mf clearance. LPS stimulation induces higher levels (~10-fold in
multiple experiments) of IL-12 from macrophages of C.D2 mice than from BALB/c
macrophages. The two strains are congenic for the Ity/Bcg/lsh locus, which is
controlled by the expression of the endo-lysosomal transporter NRAMP-1. These
enhanced levels of IL-12 induction in C.D2 macrophages were not associated
with any reduction of nitrite accumulation. However, when the rate of mf
clearance was compared between BALB/c and C.D2 mice, no significant
differences could be seen. Thus, the rate of mf clearance does not correlate
with the levels of IL-12 induced. Together, our findings show that compromised
iNOS/NO induction in macrophages appears to be more important in causing
delayed clearance of microfilaraemia, rather than the precise cytokine pattern
of the T cell responses seen. Enhancement of T cell memory in vivo by pentoxifylline We have shown earlier that the presence of
pentoxifylline (PF) during priming of human CD4 T cells in vitro against
alloantigenic target cells inhibits the primary proliferative T cell responses
but enhances secondary proliferative responses and reduces the frequency of
apoptosis in activated T cells. 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 PF can induce any physiologically relevant
modulation of immune T cell memory in vivo. Antigen-recalled T cell
proliferative responses were short-lived in mice immunized with a single dose
of OA in PBS, so that by day 60 post-immunisation no response could be
detected. In contrast, if the mice were treated daily with PF from day -2 to
day +5 of immunization, T cell responses could be recalled very efficiently
even at day 60 post-immunization. CD4 T cells produce alternate packages of
cytokines; - ‘Th1’ cytokines such as interferon-gamma (IFNg) and Th2
cytokines such as interleukin (IL)-4, IL-5 or IL-10. Treatment with PF from
day -2 to day +5 of immunization did not alter the ratio of IFNg (a Th1
cytokine) to IL-10 (a non-Th1 cytokine) in the resultant T cell responses two
weeks later. Thus, Th1 and Th2 cytokine patterns of T cell immune responses in
mice are not altered by PF. PF inhibits induction of
primary T cell proliferation as well as T cell activation-induced cell death (AICD)
in vitro. We therefore tested the effects of PF on T cell activation and AICD
in vivo by using the well-characterized model of superantigen-mediated
activation and AICD of both CD4 and CD8 T cells bearing certain T cell
receptor (TCR)-Vb elements. We used the MHC-matched mouse strains CBA/J and
CBA/CaJ differing in their mls superantigens. CBA/J mice (mls-stimulator)
express the endogenous retroviral mls-1 superantigen encoded by Mtv-7, while
CBA/CaJ (mls-responder) do not. The Mtv-7 superantigen causes deletion of CD4
and CD8 peripheral T cells expressing appropriate TCR-Vb elements, notably
TCR-Vb6. We injected mls-stimulator splenic APCs
intraperitoneally (i.p.) into mls-responder mice to induce deletional
tolerance of mls-1-reactive T cells. PF treatment during this period protected
TCR-Vb6-bearing T cells from deletional tolerisation. To examine this issue
further, cell transfers were also done in the reverse direction. Thus,
mls-1-responder T cells were transferred into mls-1-stimulator recipients with
or without PF treatment. Again, the frequency of TCR-Vb6-bearing cells was
higher in PF-treated mice than in untreated mice on day 7, in both CD4 and CD8
T cells. The very high frequencies of TCR-Vb6-bearing cells achieved from
small numbers of input donor cells in PF-treated recipients indicated that PF
may not inhibit T cell proliferation in vivo. Next, input cells from
mls-1-responder mice were marked with the fluorescent dye CFSE before being
transferred into mls-stimulator recipients. At 24 h post-transfer, CFSE-labeled
TCR-Vb6-bearing cells could be detected in mls-responder CBA/CaJ recipients as
well as in CBA/J recipients with or without PF treatment. However, these
TCR-Vb6-bearing cells had proliferated by 72 hours to the point of losing the
fluorescein label irrespective of PF treatment. By 7 days post-transfer, there
was significant reduction in the frequency of TCR-Vb6-bearing cells in the
PBS-treated recipients, while these cells persisted at virtually unchanged
levels in PF-treated mice. Thus, while PF inhibited TCR-Vb6-bearing T cell
loss in vivo, it did not significantly slow down their activation-induced
proliferation. PF did not modify the induction of cell-surface activation
markers such as CD95L, CD44, CD69 and CD122 on TCR-Vb6-bearing T cells upon
injection into mls-stimulator mice. Thus, PF treatment early during immunization generates
longer lasting T cell memory in vivo by inhibiting AICD-mediated T cell loss
rather than inhibiting T cell activation per se. As a PDE inhibitor, PF
enhances the cAMP levels induced in cells during activation. We tested if cAMP-dependent
signaling pathways are involved in enhancing T cell memory by treating mice
with either PBS, PF or dibutyryl cAMP, a cell-permeable functional analogue of
cAMP, early during immunization. The recall responses from such mice
demonstrate that dbcAMP does enhance T cell immune memory in vivo, making it
very likely that the T cell memory-enhancing effect of PF is due to increased
cAMP levels by PDE inhibition. Thus, the cAMP-dependent signaling pathway,
which is critical for long-term neural memory, can also contribute to T cell
memory. Our data identify a novel class of immunological adjuvants for
vaccination. PF is commonly used clinically, and its doses used here are
within the clinical range. The extensively analyzed cAMP-dependent pathways
should now reveal other more efficient potential enhancers of immune memory. Publications Original peer-reviewed articles 1. Raman VS, Bal V, Rath S and George A (2000)
Ligation of CD27 on murine B cells responding to T-dependent and T-independent
stimuli inhibits the generation of plasma cells. J Immunol 165:6809-6815. 2. Pasare C, Mukherjee P, Verhoef A,
Bansal P, Mendiratta SK, George A, Lamb JR, Rath S and Bal V (2001) T cells in
mice expressing a transgenic human T cell receptor b chain get positively
selected but cannot be activated in the periphery by signaling through T cell
receptor. Int Immunol 13:53-62. Reviews/Proceedings 1. Rath S (2000) Riding a tiger: Science and
craft in vaccine design. In: New millennium lectures: celebration of science
(Indian Science Congress 2000, Pune) CSIR, New Delhi and ISCA, Kolkata,
123-132. |