12
International Congress of Immunology 2016
Abstract Book
the suppressive function of human nasopharyngeal carcinoma
(hNPC) derived exosomes and to significantly limit the growth of
xeno-transplanted hNPC tumors in immuno-deficient mice that
were previously reconstituted with a human immune system.
1582
Toll-like receptor 9 is required for the maintenance of
CD25
+
FoxP3
+
CD4
+
T
reg
cells during
Listeria monocytogenes
infection
Dolina, J., Lee, J., Schoenberger, S.
La Jolla Institute for Allergy and Immunology, SanDiego, United States
It has long been appreciated, but not understood, that the
CD8
+
cytotoxic T lymphocyte (CTL) dependence on CD4
+
T cell
help (T
h
) is conditional; needed for some immunogens but
not others. One explanation for this phenomenon envisions
T
h
requirement as an intrinsic property of the pathogen itself
rather than its introduction to the immune system. Here we
show that dependence of the optimal CD8
+
T cell response to
Listeria monocytogenes
(
Lm
) on CD4
+
T cells is
a function of the immunogen dose used for priming, with low
dose
Lm
(LD; 50 or 10
3
CFU WT or Δ
ActA
, respectively) inducing
a primary antigen-specific CTL response profoundly dependent
on CD4
+
T
h
cells while that induced by high dose
Lm
(HD; 4×10
3
or 10
6
CFUWT or Δ
ActA
, respectively) is significantly inhibited by
CD4
+
CD25
+
FoxP3
+
regulatory T cells (T
reg
). The T
h
-independence
of HD immunization is not overcome by additional antigen but
instead involves the inflammatory response to more bacteria.
Evaluation of various TLR pathways as the relevant sensing
mechanism showed that TLR2 is required for CTL responses to
LD immunization, and that HD immunization in the absence
of TLR9 results in a simultaneous loss of CD25
+
FoxP3
+
CD4
+
T
reg
cells and increase in conventional CD4
+
T
h
cells and CTLs. Our
data thus reveal that the CTL response to the same pathogen
is determined by distinct roles for CD4
+
T cells as helpers versus
regulators based on immunogen dose and demonstrate a
previously undescribed role for TLR9 in the regulation of CD4
+
T
h
and T
reg
cells.
2281
High-content cytotoxic assays reveal the biological activity
of pathological and therapeutical cytotoxic T lymphocytes
Guipouy, D.
1,2,3,4
, Gertner-Dardenne, J.
4
, Belmonte, N.
4
, Dupré, L.
1,2,3
1
Centre de Physiopathologie de Toulouse Purpan, Inserm UMR
1043, Toulouse, France,
2
Université Toulouse III Paul Sabatier,
Toulouse, France,
3
CNRS, UMR 5282, Toulouse, France,
4
TxCell,
Valbonne-Sophia Antipolis, France
The killing efficiency of cytotoxic T cells is usually evaluated by
measuring the percentage of dead target cells resulting from a
4-hr coincubation with an excess of cytotoxic T cells. However,
the conventional assays fail to capture the ability of these cells to
sustain killing over prolonged time. We have recently developed
a combination of flow cytometry- and microscopy-based assays
to provide kinetic measurements of target cell elimination by
human CD8
+
T cells (Vasconcelos
et al
., Cell Rep. 2015,11:1474).
We here developed these assays further to assess in depth the
cytotoxic activity of
T cells in two clinically-relevant settings: i) CD8
+
T cells isolated
from Wiskott-Aldrich syndrome (WAS) patients, ii) antigen-
specific type-1 regulatory CD4
+
T cells (Ovasave
®
, TxCell), which
are currently tested in refractory Crohn´s Disease patients
(Phase IIb). Although WAS CD8
+
T cells were able to kill target cells in 4hr, their ability to sustain
killing over 24hr was strongly impaired. The defect of WAS CD8
+
T cells to control target cells over time was linked to an inability
to assemble stable synapses, resulting from abnormal actin
cytoskeleton organization and LFA-1 activation. Our assays also
revealed that a potential mechanism of action of therapeutic
regulatory T cells relied on the elimination of myeloid cell
via
a cytotoxic mechanism. Interestingly, these unconventional
cytotoxic
T cells developed a robust cumulative killing activity over
24hr. Our study highlights the advantage of applying flow
cytometry- and microscopy-based assays to characterize, with
high sensitivity, the kinetics of the cytotoxic activity of clinically-
relevant T lymphocytes.
Allergy 1
1245
The Nedd4-2-Ndfip1 axis is essential to curtail mast cell-
dependent IgE-mediated anaphylaxis
in vivo
Yip, K.H.
1
, Kolesnikoff, N.
1
, Hauschild, N.
1
, Lopez, A.F.
1,2
, Galli, S.J.
3
,
Kumar, S.
1,2
, Grimbaldeston, M.A.
1,2
1
Centre for Cancer Biology, University of South Australia and SA
Pathology, Adelaide, Australia,
2
University of Adelaide, School of
Medicine, Adelaide, Australia,
3
Stanford University, Departments of
Pathology and of Microbiology and Immunology, Stanford, United
States
Cross-linkage of the IgE receptor (FcεRI) by specific antigen
ligation on mast cells (MCs) plays a critical role in the
pathological process of IgE-dependent allergic disorders, such
as anaphylaxis and asthma. Restraint of intracellular signal
transduction pathways that promote release of MC-derived pro-
inflammatory mediators is necessary to dampen activation and
restore homeostasis. In the course of studying the molecular
basis regulating IgE-mediated MC function, we discovered
that the ubiquitin ligase, Nedd4-2, is a major gatekeeper of
MC activation and the adapter molecule Ndfip1 (Nedd4 family
interacting protein 1) has a role in this process.
Herein, we show that loss of Nedd4-2 or Ndfip1 activity in MCs
causes an increase in themagnitude and duration of IgE-induced
pro-inflammatory mediator release
in vitro
. A closer inspection
of the signalsome activities of Fc
ε
RI engagement in
Nedd4-2
-
/-
and
Ndfip1
-/-
MCs compared to the wild-type counterparts,
revealed that p-Syk polyubiquitination by Nedd4-2/Ndfip1
proximal to FcεRI aggregation was disrupted, leading to
exacerbated calcium mobilization and prolonged propagation
of multiple downstream signaling events including those of the
ERK1/2 and NF-κB pathways. The biological significance of these
investigations is highlighted further in an IgE-mediated MC-
dependent passive cutaneous anaphylaxis mouse model that
employed the adoptive transfer of
Nedd4-2
-/-
and
Ndfip1
-/-
MCs