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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