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In-Depth Information
transfer colitis, Haribhai and colleagues could demonstrate that prevention
of the disease, as well as its cure, could be fully achieved only in the presence
of both Treg and iTreg cells
[58]
. Whether this reflects differences in sup-
pressive mechanisms used by the two Treg cell types that need to synergize
for full protection or differences in their TCR specificities that complement
each other
[59]
remains to be determined.
Suppressive activity of Treg cells was first demonstrated
in vitro
in a 3-day
proliferation assay
[29,60]
. This assay is still widely used to assess Treg activ-
ity despite its limited reflection of the overall capacity of Treg cells
in vivo.
In its original configuration, Treg cells and CD4
+
or CD8
+
responder T cells
(Tresp) are cocultured with APC and stimulated polyclonally through the
addition of anti-CD3 antibodies. Suppression of Tresp cell proliferation is
then evaluated by incorporation of [
3
H]TdR into the DNA during the last
4 to 12 h of the assay. Since its initial description, this assay has been var-
ied in several ways to incorporate new findings and address new questions
regarding the nature and the mode of action of the molecules instrumental
in this suppression process, including variation of responder cells and APC
or replacing APC altogether with antibody-coated beads, use of a specific
antigen instead of anti-CD3 antibodies, labeling of Tresp with CFSE and
other cell proliferation dyes, or determining the inhibited upregulation of
activation markers such as CD154 and CD69 on Tresp cells instead of their
proliferative response
[61,62]
. Separating Tresp and Treg cells by a semi-
permeable membrane abrogated suppression and thus led to the notion
that cell contact was an important prerequisite, whereas soluble mediators
with known immunosuppressive activity, such as IL-10 and TGF-β, seemed
dispensable
[29,60]
. In subsequent studies CTLA-4 and IL-2R were identi-
fied as two main molecules involved in cell-contact-dependent suppres-
sion by Treg cells. CTLA-4 has been shown to be constitutively expressed
by mouse and human Treg cells, whereas it is upregulated in Tconv cells
only after activation
[63,64]
. Combined with the finding that CTLA-4-defi-
cient mice develop a lymphoproliferative disease that results in multiorgan
tissue destruction and death of the animals by 3 to 4 weeks
[65]
, CTLA-4
became one of the prime targets of Treg research with regard to suppressive
mechanisms. Yet, things became more complicated when Treg from CTLA-4
knockout (KO) animals were shown to be equally suppressive to wild-type
(WT) Treg in a standard
in vitro
suppression assay
[66]
. These results are best
explained by the highly activated state of Treg cells isolated from CTLA-4
KO animals with ongoing inflammation, but are still in conflict with subse-
quent studies by Wing and colleagues, who generated Treg-specific CTLA-4
KO mice. These mice also succumb to lethal systemic lymphoproliferative
disease, despite normal Treg cell numbers and CTLA-4 expression in Tconv
cells. However, Treg cells taken from these mice show a significant reduc-
tion in their suppressive activity compared to WT Treg cells
in vitro
as well
as
in vivo.
Thus, loss of CTLA-4 clearly renders Treg cells less effective
[67]
.
CTLA-4-mediated suppression has been proposed to happen mostly via
interaction with its ligands CD80/CD86 on APC, which leads to downregu-
lation of these costimulatory molecules and thus reduced capacity of the
APC to activate Tconv cells
[67]
. Simultaneously, this interaction can induce
the expression of indoleamine 2,3-dioxygenase (IDO) in DC, the enzyme
that catalyzes tryptophan degradation and production of kynurenines,
250
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