|
Biology
of T lymphocytes |
| Principal Investigator : Vineeta Bal
Project
Associates/Assistants Ph D Students
Collaborators Characterisation
of signals received by T lymphocytes and antigen presenting cells during
activation is the theme of research. The objectives of the project are to (i)
study the role of inducible nitric oxide synthase (iNOS) in T lymphocyte
responses, (ii) characterise the role of T cells as antigen presenting cells
for CD4 and CD8 T cells and (iii) analyse toll-like receptor mediated
signalling in macrophages. 1.
To study the role of inducible nitric
oxide synthase in T lymphocyte responses Last
year we reported early in vitro studies on T cells from iNOS-/-
mice, which indicated that the absence of iNOS may be correlated to cell
survival and immune memory. This was confirmed during the last year with
various approaches. WT
or iNOS-/-
mice were immunized with a protein immunogen, maleylated ovalbumin (maleyl-OVA),
on alum. Six weeks post-immunization, the T cell proliferative responses
recalled in culture with maleyl-OVA were greater in the iNOS-/-
mice than in WT counterparts which was a result of increase in the clonal
frequency of maleyl-OVA-specific T cells from iNOS-/-
mice and not due to differences in the burst size between WT and iNOS-/-
T cells. Antigen-specific CD4 and CD8 memory responses were further
characterised by using immunisation of mice with MHC class I and class II
restricted OVA-derived peptides. Using flowcytometry, peptide-recallable cells
which showed activated status (CD44hi)
and were producing intracellular interferon-gamma (IFN-g) were scored from
immune mice 42 days post-immunisation. The data showed that both CD4 and CD8 T
cells from iNOS-/-
mice were present in larger numbers than those from WT mice. Longer
survival of antigen-primed T cells in vivo in unimmune iNOS-/-
mice due to environmental exposure to antigens was also indirectly
demonstrated by comparing their numbers and activation status with unimmune WT
mouse T cells. Both CD4 and CD8 cell numbers were marginally higher in iNOS-/-
mice and many more of these showed activated phenotype (CD44hi
or CD62Llo). The
next issue was whether iNOS in T cells or in APCs is contributing to the
enhanced memory response seen in these mice. Using iNOS-/-
T cells as responders and MHC mismatched iNOS+/+
cells as APCs, and vice versa, we clearly showed that endogenous iNOS
produced in T cells is necessary for their longer survival. The allo-priming
was done both in vitro and in vivo and gave the same results. By
Western blot we also demonstrated that iNOS is indeed present in T cells from
WT mice. Since
longer survival of T cells in vivo is likely to be a result of decrease
in their susceptibility to death, we investigated the role of apoptosis in
immune memory in iNOS-/-
and WT T cells. Activated T cells are known to undergo activation induced cell
death (AICD). T cells also undergo trophic factor withdrawal death (TSWD) with
IL-2, IL-7 and IL-15 as known trophic factors in this case. Using T cell
blasts generated from WT, iNOS-/-
or CD95-deficient B6.lpr mice and providing appropriate stimuli for the
induction of AICD or TSWD, we scored death in these cells post-activation. The
DNA-binding dye Hoechst 33342 was used to determine proportions of cells with
apoptotic nuclei by microscopy. While T cells from B6.lpr mice were
resistant to AICD, they were as susceptible as WT T cells to TSWD. In
contrast, iNOS-/-
T cells were resistant to TSWD whereas they were as susceptible as WT T cells
to AICD. These data clearly indicated that TSWD plays a major role in the
death of T cells post-activation. Absence of which makes cell survive much
longer thereby providing enhanced memory response. The role of TSWD in immune
memory was confirmed in two different ways using T cells from B6.lpr
mice. In a functional assay B6.lpr T cells were allo-primed in presence
or absence of peroxynitrite scavenger MnTBAP, and mopping of nitric oxide
during priming resulted in enhanced secondary or memory response in these T
cells. Secondly, addition of MnTBAP to T cell activating cultures resulted in
decreased death of B6.lpr T cells by TSWD. In
order to dissect molecular pathways associated with longer survival of iNOS-/-
T cells post-activation, we looked at the role of bcl family members by
Western blotting. Cell extracts from activated iNOS-/-,
WT or MnTBAP-treated WT T cells cultured under various conditions showed that,
by 12h after IL-2 withdrawal, WT T cells ceased to show expression of either
Bcl-2 or Bcl-xL, in contrast to cells maintained in the presence of IL-2.
However, if the WT T cells were treated with MnTBAP, or if iNOS-/-
T cells were subjected to IL-2 withdrawal, substantial levels of Bcl-2 and
Bcl-xL continued to be present. In
previous years we had reported that human T cells also respond better when
primed in presence of MnTBAP. We have now shown that this longer survival is
also due to inhibition of TSWD in them. Thus,
we have comprehensively shown that iNOS in T cells contributes to the post
activation death of T cells in vivo by downmodulating anti-apoptotic
members of the bcl family such as Bcl-2 and Bcl-xL. In the absence of
iNOS in T cells, they persist for much longer period after activation thereby
contributing to enhancement of immune memory. 2.
To characterise the role of T cells as antigen presenting cells for CD4 and
CD8 T cells Over
the years we have been reporting the consequences of antigen presentation by
activated human T cells on responder T cells. Due to various limitations
imposed by the human allo-recognition system, we decided to look at the
ability of mouse T cells as APCs. We used three types of transgenic mice, one
expressing TCR specifically recognising an OVA-derived peptide presented by I-Ab
(OTII-TCR-Tg), second recognising a peptide derived from lymphocytic
choriomeningitis virus in association with Kb (p14-TCR-Tg) and the third one
expressing I-Ab on T cells (JLA). We used ex vivo naive and
activated T cells as APCs and compared their ability with dendritic cells (DCs)
as professional APCs. Our results show that both CD4 and CD8 TCR-Tg T cells
can be primed very well in vitro by peptide-pulsed DCs as APCs. These
primed T cells respond vigorously to a secondary challenge by splenic APCs and
produce secondary cytokines such as IFN-g. When either naive T cells or
activated T cells or JLA-Tg T cells are used as APCs for priming in vitro,
priming efficiency is very poor as judged by a recall response to peptide
pulsed splenic APCs. In addition, these weakly primed T cells produce very
little IFN-g indicating that they are anergised. Thus these data confirm and
extend our earlier findings in the human system showing that both CD4 and CD8
T cells can be turned off when antigens are presented by T cells as priming
APCs. Both naive and activated T cells used as APCs provide the anergising
signal to the responder T cells. These finding have important implications in
T-APC mediated anergy in vivo as a potential downmodulatory event in an
ongoing infection scenario. 3.
To analyse toll-like receptor mediated signalling in macrophages We
have used C3H.HeJ (HeJ) and C3H.HeN (HeN) strains of mice. While C3H.HeN are
normal, C3H.HeJ show natural mutation for toll-like receptor (TLR) 4 because
of which C3H.HeJ mice do not respond to LPS, a ligand for TLR4. Our
preliminary work shows that S.digitata microfilarial extract can
stimulate peritoneal exudate cells (PECs) from HeN mice to produce higher
levels of nitrites as compared to those from HeJ mice in a dose dependent
fashion. S.digitata mf are reported to be free of the endosymbiont
bacteria Wolbachia and LPS derived from it. However, these mf are
collected from the slaughter house with a potential for contamination of mf
with LPS, and hence we repeated the experiment in presence polymixin B. We
observed that polymixin B brings down nitrite production significantly from
HeN macrophages, however, nitrite production from HeJ macrophages is unaltered
by polymixin B. In order to circumvent LPS related problems, we also used a
purified antigen of mf, AgW, to stimulate PECs from HeJ and HeN strains. AgW
vigorously stimulates HeN macrophages to produce nitric oxide read out as
nitrites. This is again partially inhibited in presence of polymixin B.
However, AgW cannot stimulate HeJ macrophages to produce nitrites above
background level. This seems to suggest that in the absence of TLR4 AgW cannot
stimulate macrophages to produce nitrites. We
next looked at the ability of activated macrophages to kill microfilaria in
vitro in these two strains in presence or absence of LPS. While in the
absence of LPS both HeN and HeJ macrophages failed to kill mf even at 72
hours, addition of LPS in cultures resulted in efficient killing of mf in
LPS-responder HeN strain macrophages. These data clearly demonstrate that
presence of LPS and hence signalling through TLR4 is significantly responsible
for mf killing. It is still not clear whether stimulation through any TLR
would bring about enhanced mf killing or this is peculiar to LPS.
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. |