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

• To examine the requirement for Atg5 in the initiation of immune responses in vivo

Aim

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

To isolate the effect of Atg5 deficiency on cDcs

Atg5 -/- chimeric miceWT mice

HSV-1 infection

cDc purification

day 3 post infection

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 flox/flox

DC-Atg5 -/-

HSV-2 Intravaginal infection

Isolate lymph node on day 7 and CD4+T cells purification

+ WT APCs

+ 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) +

naïve OVA-specific OT-I cells

(DCs + OVA) +

naïve OVA-specific OT-II cells

To examine the presentation of apoptotic cell-associated antigen

WT or Atg5 -/- splenic cDCs+

Irradiated OVA-loaded MHC II-deficient 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