851
and pathogen-derived factors on DCs to find out which reagent can
efficiently induce tolDCs. BMDCs were stimulated with those factors
and the expressions of co-stimulatory, co-inhibitory, and MHC
molecules on BMDCs were observed. In addition, we investigated
cytokine profiles of BMDCs by ELISA to further estimate tolerogenic
potential of BMDCs. Notably, treatment of inflammatory cytokine,
IFN-γ, up-regulated the expression of inhibitory molecules (PD-L1
and PD-L2) on DCs. Another interesting finding was that treatment
with one of the inhibitors for glycogen synthase kinase 3 beta (GSK-
3β) slightly up-regulated PD-L1.
Moreover, upon stimulation with lipopolysaccharide (LPS), the
GSK-3β inhibitor-treated DCs showed significantly increased IL-10
production. Finally, we performed functional assay using OT-I and
OT-II cells to test the abilities for antigen uptake, processing and T
cell priming of induced tolDCs. Through our investigation, we could
take another step for searching tolerogenic agent and establishing
protocols for generating tolDCs, which can be further applied into
clinical trialswhere immune suppression is needed.
Histamine contributes to the resistance against tick-blood-
feeding during the re-infestation
Ohta,T.
1
,Yoshikawa,S.
1
,Ishiwata,K.
2
,Yamaji,K.
2
,Okayama,N.
1
,
Yamanishi, Y.
1
, Kanuka, H.
2
, Watanabe, N.
3
, Ohtsu, H.
4
, Karasuyama, H.
1
1
TokyoMedicalandDentalUniversity,DepartmentofImmune
Regulation,Tokyo,Japan,
2
JikeiUniversitySchoolofMedicine,
DepartmentofTropicalMedicine,Tokyo,Japan,
3
JikeiUniversity
SchoolofMedicine,DepartmentofAllergology,Tokyo,Japan,
4
Tohoku
University,DepartmentofEngineering,Miyagi,Japan
Ticks transmit a variety of pathogens from reservoir host to humans
and animals during the tick-blood-feeding, leading to tick- borne
infectionssuchaslymedisease.Somestudieshavereportedthattick-
infested animals acquired resistance against tick- blood-feeding and
furthermoreprevent transmissionof pathogens fromtick to animals.
We previously reported that basophils infiltrate around the tick-
bite-sites during the re-infestation but not primary infestation, and
basophil-depletion before re-infestation can abolish the resistance
againsttick-blood-feeding.Moreover,adoptivetransferexperiments
elucidated the importance of Ig-Fc receptors on basophils for the
resistance against tick-blood-feeding.These findings suggested that
basophil-activation through the Ig-Fc receptors are important for
the resistance against tick-blood-feeding during the re-infestation.
Basophils are well known for their release inflammatory molecules
such as histamine and protease from theirs secretary granules
in response to IgE/antigen stimulation. We examined whether
histamine is involved in resistance against tick-blood-feeding
using histamine (
HDC
)-deficient mice. We confirmed that immune
reactions such as the levels of tick antigen reactive IgE, basophil-
infiltration and their-activation in tick-feeding-sitewere comparable
betweenwild type and
HDC
-deficientmiceduring the re-infestation.
However,
HDC
-deficient mice failed to develop resistance against
tick-blood-feeding. These findings implied that histamine released
by basophils upon IgE plus tick antigens is important for the
resistance to tick-blood-feedingduring the re-infestation.
Blood gene expression profiles from distinct outcomes of
infection with Leishmania infantum
Gardinassi,L.G.
1
,RochaGarcia,G.
1
,CostaSilva,V.
2
,Costa,C.H.N.
2
,de
MirandaSantos,I.K.F.
1
1
UniversityofSaoPaulo-RibeiraoPretoMedicalSchool,Biochemistry
andImmunology,RibeiraoPreto,Brazil,
2
Federal UniversityofPiauí/
NatanPortellaTropicalDiseasesInstitute,Teresina,Brazil
Visceral leishmaniasis (VL) canbe lethal if left untreated; however, the
majority of human infections with the etiological agent
Leishmania
infantum
,areasymptomatic.UsingBeadChipmicroarraytechnology
(Illumina), we investigated the profiles of gene expression from
blood of VL patients, patients that received therapy, asymptomatic
individuals and controls. Our data demonstrate that each group
included in the study presents a unique transcriptional signature.
By employing computational analysis based on diverse functional
methods as differential gene expression, gene set enrichment
analysis, weighted gene co-expression network analysis and
immune cell deconvolution, our findings support that VL patients
present transcriptional profiles associated to an activation of T
lymphocytes via MHC class I. Of interest, VL patients also presented
negative associations with transcriptional modules related to cell
adhesion and chemotaxis, monocytes, neutrophils and B cells.
Treated patients presented with a mixed transcriptional profile,
sharing several features of untreated patients or healthy individuals.
This group of study presented a positive regulation of cell cycle and
proliferation of T cells, as well as transcriptional activity correlated
with a modulation of cell adhesion and chemotaxis and the Notch
signaling pathway. Asymptomatic and uninfected individuals
present similar patterns of gene expression. Nevertheless,
asymptomatic individuals present subtle particularities, such as a
strong associationwith type I interferon
response.Weconclude that
VL patients present dysfunctions on several compartments of the
immune response and suggest that an efficient global regulation of
the immune response is associated with protection to development
of clinical symptoms.
Financialsupport:FAPESP
A multi-color Natural Killer-cell mediated cytotoxicity
detection of tumor cells using fluorescence and direct cell
imaging
Sundararaman,S.,Roen,D.,Karulin,A.,Caspell,R.,Lehmann,P.
CellularTechnologyLtd,ShakerHeights,UnitedStates
The most essential role of effector immune cells such as CD8+ cells
andNatural Killer (NK) cells is to identify and lyse target cells. NK Cell -
and Antibody Dependent (ADCC) Cell - Cytotoxicity has traditionally
been assessed by the release of radioactive Chromium from
target cells following lysis. These assays are laborious and require
substantialquantitiesofpatientbloodtodetect minorchangesincell
lysis.Wehave previously shown that we can achieve similar results as
the Chromium Release Assay by the direct imaging of fluorescently
labelled target cells and visualizing the loss of fluorescence signal
upon cytolytic activity. We showed that when performed in a 96-
well plate, the % of target cell lysis is inversely proportional to the
number of effector cells in each well. In order to further reduce the
amount of cell material required and detect the effect of NK cells
on different target cell lines we have now developed a multi-color