Anusara Daenthanasanmak 17.01.2011
description
Transcript of Anusara Daenthanasanmak 17.01.2011
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Anusara Daenthanasanmak
17.01.2011
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Autophagy is the process involving the degradation of a cell's own components through the lysosomal machinery
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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
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• To examine the requirement for Atg5 in the initiation of immune responses in vivo
Aim
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Results1. Impaired CD4+ T cell Priming by Atg5-deficient APCs
Liver cells from Atg5 -/- neonates
Atg5 -/- chimeric mice
HSV-1 intravaginal infection
CD4+ T cells isolation
+ WT APC
WT mice
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To isolate the effect of Atg5 deficiency on cDcs
Atg5 -/- chimeric miceWT mice
HSV-1 infection
cDc purification
day 3 post infection
CD4+
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To examine the ability of WT T cells primed by Atg5 -/- APCs in vivo
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To provide evidence for the in vivo role of autophagic machinery in antigen presentation by cDcs
CD11c-Cre Atg5 flox/flox
DC-Atg5 -/-
HSV-2 Intravaginal infection
Isolate lymph node on day 7 and CD4+T cells purification
+ WT APCs
+ HSV-Ag
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Lethal dose of HSV-2
DC-Atg5 -/-
DC-specific Atg5 -/- mice fail to prime antiviral Th1 cells and succumb to HSV-2 infection
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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
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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
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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
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To examine the importance of autophagy in presentation of cytosolic Ag
Infect DCs with OVA-expressing Listeria monocytogenes
(DCs + OVA) +
naïve OVA-specific OT-I cells
(DCs + OVA) +
naïve OVA-specific OT-II cells
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To examine the presentation of apoptotic cell-associated antigen
WT or Atg5 -/- splenic cDCs+
Irradiated OVA-loaded MHC II-deficient splenocytes
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To examine the kinetics and extent of peptide loading onto MHCII with a pulse-chase analysis
Localization of phagocytosed Ag and MHC II
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Impaired phagolysosomes of
the Atg5 -/- DCs
Kinetics of lysosomal and phagosomal pH in WT Atg5 -/- DCs
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To examine if there is a defective delivery of lysosomal protease to the phagosomes
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• 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
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