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Evaluation of immunosuppressive function of regulatory T cells using a novel in
vitro cytotoxicity assay
#MMPMID25908962
Zhang L
; Manirarora JN
; Wei CH
Cell Biosci
2014[]; 4
(?): 51
PMID25908962
show ga
Naturally occurring regulatory T cells (Tregs) play a pivotal role in the
maintenance of self-tolerance due to their intrinsic immunosuppressive activity.
Currently, a number of human clinical trials are being conducted to investigate
the roles of Tregs in treating various immune-mediated disorders. Traditionally,
the suppressive activity of Tregs is measured using either a thymidine
incorporation assay, which is a radioactive assay; or CFSE based flow cytometry
assay, which requires a relatively large number of cells. Consequently, there is
an increasing need to develop novel alternative bioassays that can characterize
various aspects of the immunosuppressive function of Tregs in vitro. In this
study, using murine clonal CD8(+) T cells specific for an islet antigen as
responder T cells, we first established a novel, sensitive and quantitative in
vitro luminescence based cell viability assay to measure cytotoxicity. Then we
used this assay to measure if Tregs could inhibit the cytotoxicity of CD8
effector T cells. This assay does not involve the use of radioisotopes and only
needs relatively low number of Tregs. Since normally Tregs only constitute 5-10%
of peripheral CD4(+) T cells, this advantage is noteworthy compared with other
methods. With the assay we developed, we demonstrated that regulatory T cells
(Tregs) could inhibit the antigen-specific killing of an adherent target cell
monolayer by the CD8(+) cytotoxic T cells. We observed more inhibition when Tregs
and CD8 killer T cells were incubated during the in vitro activation
(stimulation) stage of the cytotoxic T lymphocytes (CTL) than when they were
added later at the start of the effector phase. Interestingly, Tregs from B6 mice
demonstrated higher suppression of CD8(+) T cell killing than Tregs from NOD
mice. Moreover, IL-2/anti-IL-2 mAb complexes induced expansion of Tregs in vivo,
as well as enhancing the Treg's suppressive activity per cell. Therefore, this
novel non-radioactive, luminescence based cytotoxicity assay mediated by clonal
islet antigen-specific CD8 T cells can be used to measure, characterize, and
quantitate the immunosuppressive activity of natural Tregs, representing a useful
approach to characterize the functions of Tregs in the setting of autoimmune
diseases and to elucidate the mechanisms for Treg cell-mediated immunoregulation.
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