Transcript To examine if HSV-infected WT and Atg5

Anusara Daenthanasanmak
17.01.2011
Autophagy is the process involving the degradation of a
cell's own components through the lysosomal machinery
In vitro
• Recent studies suggested the involvement of autophagy in MHC II presentation
of intracellular antigen
• By using pharmacological inhibitors of the class III PI3 kinase, 3-methyladenine
(3-MA) and Wortmannin, MHC II presentation of peptides derived was shown to
be impaired in mouse macrophages and B cell line (Brazil et al., 1997)
• MHC II presentation of nuclear antigen 1 of EBV (EBVNA1) is reduced by
siRNA-mediated knockdown of Atg12
• The delivery of a MP1 antigen to the autophagosomal enhanced MHC II
presentation
• The contribution of autophagic delivery of antigens in CD4+ T cell priming in
vivo remains unclear
Aim
• To examine the requirement for Atg5 in
the initiation of immune responses in vivo
Results
1. Impaired CD4+ T cell Priming by Atg5-deficient APCs
Liver cells from
Atg5 -/- neonates
WT mice
Atg5 -/- chimeric mice
HSV-1 intravaginal infection
CD4+ T cells isolation
+ WT APC
To isolate the effect of Atg5 deficiency on cDcs
HSV-1 infection
WT mice
Atg5 -/- chimeric mice
day 3 post infection
cDc purification
CD4+
To examine the ability of WT T cells primed by Atg5
-/- APCs
in vivo
To provide evidence for the in vivo role of autophagic machinery in
antigen presentation by cDcs
CD11c-Cre
Atg5
DC-Atg5
flox/flox
-/-
Isolate lymph node on day 7
and CD4+T cells purification
+ WT APCs
HSV-2 Intravaginal infection
+ HSV-Ag
Lethal dose of HSV-2
DC-Atg5
-/-
DC-specific Atg5 -/- mice fail to prime antiviral Th1 cells
and succumb to HSV-2 infection
To examine the contribution of Atg5 in DC migration in vivo
• The ability of endogenous skin DC population to migrate to the lympnode
• 1% FITC painting
• No defects in the ability of Atg5 -/- DCs to migrate from the skin to the lymph
nodes
To examine if HSV-infected WT and Atg5 -/- DCs have similar capacity to
present antigens on MHC II
• Pulsed HSV-infected DCs with exogeneous OVA peptide
• Stimulate OT II cells
• OT II cells have similar extent of proliferation when use WT or Atg5 -/- DCs
• Similar in secretion of cytokines and no difference in the mRNA expression
Intact migration and Innate responses by Atg5 -/- DCs
To test if Atg5 is required for uptake of antigens
WT or Atg5 -/- splenic cDCs
+
OVA conjugated to pH –
insensitive fluorochrome
WT or Atg5 -/- splenic cDCs
+
Apoptotic MHC II-deficient
splenocytes labeled with the
membrane dye PKH26
cDCs do not require Atg5 for
endocytic or phagocytic uptake
of exogeneous antigens
To examine the importance of autophagy in presentation of cytosolic Ag
Infect DCs with OVA-expressing
Listeria monocytogenes
(DCs + OVA) +
(DCs + OVA) +
naïve OVA-specific OT-II cells
naïve OVA-specific OT-I cells
To examine the presentation of apoptotic cell-associated antigen
WT or Atg5 -/- splenic cDCs
+
Irradiated OVA-loaded MHC IIdeficient splenocytes
To examine the kinetics and extent of peptide loading onto MHCII
with a pulse-chase analysis
Localization of phagocytosed Ag and MHC II
Impaired phagolysosomes of
the Atg5 -/- DCs
Kinetics of lysosomal and
phagosomal pH in WT Atg5 -/- DCs
To examine if there is a defective delivery of
lysosomal protease to the phagosomes
• Antigen capture, migration, maturation and cytokine secretion by DCs is
unimpaired in the absence of Atg5
• In the absence of Atg5, DCs had a reduced capacity to process cytosolic
antigens for MHC II presentation
• Atg5 -/- DCs were impaired in ability to process phagocytosed antigen for
loading onto MHC II, due to the impaired phagosome-to-lysosome fusion
and delivery of lysosomal proteases to the phagosomes