Anusara Daenthanasanmak

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

Results 1. 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 mice WT 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