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Biology of T lymphocytes |
| Principal Investigator : Vineeta Bal Project Associates
/ Assistants Ph D Students Collaborators Analysis
of effects of pharmacological agents on the activation of lymphocytes is the
theme of research. The objectives of the project are to (i) study the role of
modulators of intracellular reactive oxygen and nitrogen species in T
lymphocyte responses, and (ii) analyse the effects of modulators of
intracellular cAMP levels on T dependent and independent B cell responses. To
study the role of modulators of intracellular reactive oxygen and nitrogen
species in T lymphocyte responses Earlier
we had reported that superoxide dismutase (SOD) mimetic, and peroxynitrite
scavenger Mn (III) tetrakis (5,10,15,20-benzoic acid) porphyrin (MnTBAP),
which protects T cells from superoxide generation enhances immune memory in
vitro and in vivo by providing protection from cell death. Since presence of
MnTBAP influences both the amount of reactive oxygen intermediates (ROI) and
reactive nitrogen intermediates (RNI) present in the cell, we undertook
experiments to find out what roles ROI and RNI play in secondary T cell
response enhancement. For this, we used human allo-recognition system in
vitro. Peripheral blood mononuclear cells (PBMCs) from HLA-mismatched
responder-stimulator donors were used. Responder T cells were primed in vitro
with irradiated stimulator PBMCs in presence or absence of various
pharmacological agents and the ability of primed T cells to respond to same
allo-PBMCs was measured by tritiated thymidine incorporation as a secondary or
memory response. Glutathione
decreases availability of ROI in the cell and does not directly affect RNI
availability. Hence we used a cell permeable analogue of glutathione -glutathione
monoethyl ester- during T cell priming. Unlike MnTBAP which enhances immune
memory, priming in presence of glutathione monoethyl ester did not enhance the
secondary response indicating that the role of MnTBAP as SOD and peroxidase
mimic may not be significant in immune memory. Nitric oxide synthase (NOS)
enzymes are the major contributors to the RNI in various cells. There are at
least three types of NOS present in the body and we used a pan-NOS inhibitor
L-NMMA during allo-priming. The T cells primed in presence of L-NMMA showed
enhanced secondary response similar to one observed with MnTBAP. In order to
identify which specific NOS might be contributing to the observed effect, we
first used NOS2 (iNOS)-specific inhibitor aminoguanidine (AG) during T cell
priming and it showed the same effect as observed with pan-NOS inhibitor.
These data clearly indicated that NOS2 inhibition during T cell priming is
responsible for enhanced secondary response and immune memory. We
had reported earlier that anti-CD3 mediated activation induced T cell death is
retarded in presence of MnTBAP. We have used AG and L-NMMA in a similar assay
and find that decrease in available RNI during T cell activation inhibits post
activation T cell death. We
have also analysed the effects of ROI and RNI on mouse T cells. Presence of
MnTBAP during allo-priming of mouse T cells results in enhanced secondary
response as seen in human T cells. We have also validated the efficacy of
MnTBAP to enhance immune memory when used during protein immunisation of mice.
Since all these data suggest a major role for RNI in immune memory, we used
NOS2null mice for dissecting the role of NOS2 in immune memory. The
ability of T cells from NOS2null (H-2b) mice to respond in vitro to allo-antigen
presenting cells (APCs) from BALB/c (H-2d) mice was compared to C57BL.6 (Wild
type, WT) mice. Primed NOS2null and WT cells were harvested and labeled with
CFSE, a dye used routinely for monitoring cell divisions by flowcytometry.
Irradiated BALB/c stimulators were used for secondary recall and the number of
dividing cells was monitored from the two groups over 72-96 hours by
flowcytometry. Our data show that T cells from NOS2null mice are present in
larger numbers as compared to those from WT mice at the end of priming.
However, the rate of cell division does not appear to be different between the
two groups of primed T cells. Thus, the data show that priming results in
larger number of cells in NOS2null mice as compared to WT mice. Whether the
increased numbers of primed cells seen is due directly to absence of NOS2 in T
cells leading to T cell survival remains to be confirmed. To
analyse the effects of modulators of intracellular cAMP levels on T dependent
and independent B cell responses. We
have extensively characterised the effects of PF on T cell activation and
death. Now we are attempting to see whether T-dependent and T-independent B
cell responses can be influenced by PF and other mediators causing increase in
intracellular cAMP levels. We
have used increasing doses of PF in splenocyte cultures stimulated with
T-independent We
next looked at the ability of PF to modulate T-dependent B cell responses. Our
preliminary results show that while B cell proliferation brought about by allo-stimulus
provided in the form of T cell help is partially inhibited in presence of PF
in a dose-dependent manner, however, there is no significant inhibition of the
antibody secretion. 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. Reviews/Proceedings 1.
Bal V (2002) Gendered science: women as practitioners and as targets of
research. Economic Political Weekly 37:5163-5167. |