Mechanisms regulating MHC II antigen presentation for improved vaccination strategies

© 2013 Dr. Jessica Michelle MoffatActivation of the adaptive immune system is critical for successful vaccination outcomes. Antigen presentation plays an instrumental role in activation of the adaptive immune response and thus vaccination success. Professional antigen presenting cells (APC) are capable of constitutive major histocompatibility complex (MHC) I and MHC II antigen presentation. The most well characterised APC are B cells, dendritic cells (DC) and macrophages. Targeting antigen to the different professional APC can dictate the resulting immune response and thus the outcome of vaccination. This Thesis focuses on the role of antigen presentation in activation of the immune system, in particular the mechanisms different APC utilise to present antigens, including protein trafficking, antigen uptake and antigen processing. Virus-like particles (VLP) are naturally occurring non-infectious immunogenic particles of viral proteins. Hepatitis B virus (HBV) small envelope protein (HBsAgS) VLP are utilised in the current HBV vaccine. In Chapter Three we demonstrate that HBsAgS VLP can be utilised to generate adaptive immune responses with presentation of both MHC I and MHC II antigens observed following administration in vitro and in vivo. Despite the inhibition of interferon α (IFNα) secretion by plasmacytoid DC (pDC) in vitro, DC are capable of generating specific CTL responses to epitopes within HBsAgS VLP. In Chapter Four we investigate targeting of antigen to surface receptors in order to generate adaptive immunity. We investigate bone marrow stromal cell antigen 2 (BST-2) as a targeting receptor for delivery of antigen to the MHC I and MHC II antigen presentation pathways. The ability of cell surface BST-2 to be internalised and trafficked to lysosomal compartments, provided evidence for BST-2 to act as a targeting receptor. Comparison of the antigen targeting ability of BST-2 to known receptors, SiglecH and DEC205, showed BST-2 targeted antigen is delivered for presentation, with better or similar efficiency than the known receptors, respectively. Importantly, the most efficient cell type at cross presenting BST-2 targeted antigens was not the pDC, which received the highest antigen load, instead the activated CD8+ conventional DC (cDC). Despite success in eliciting MHC I and MHC II antigen presentation during vaccination, a number of the mechanisms controlling these pathways are poorly understood. Ubiquitination plays a key role in regulating multiple aspects of the MHC II antigen presentation pathway. In cDC membrane associated RING-CH (MARCH) 1 is critical for the surface turnover of MHC II during the steady state. However following activation, cDC shutdown gene expression of MARCH 1, leading to increased surface expression of MHC II. The role of MARCH 1 in regulating MHC II turnover in macrophages is less defined. In Chapter Five we show that the continued expression of MARCH 1 following macrophage activation is partially responsible for the continued ubiquitination and turnover of MHC II. The role of MARCH 1 in regulating trafficking and turnover of MHC II and CD86 in APC is well characterised. However, the ubiquitination targets of the other MARCH family members remain unknown. In Chapter Six we adapt a recently established protocol for plasma membrane proteomics analysis by mass spectrometry to identify known targets of MARCH 1, confirming that this protocol can be utilised to identify targets of the other MARCH family members. Additionally, the individual MARCH family members have unique gene expression profiles in an array of immune cells, highlighting the need to target specific cell types for proteomics analysis. Utilising techniques in molecular, cellular, biochemical and advanced proteomics analysis this Thesis has advanced the fields understanding of the activation of the adaptive immune response through antigen presentation. In particular this Thesis has outlined the mechanisms regulating MHC II antigen presentation for improved vaccination strategies