DNA vaccination is a novel means of expressing antigens in vivo for the generation of humoral
and cell-mediated immune responses. DNA vaccines elicit protective immunity in mice; however,
DNA vaccination in outbred species requires optimisation. Dendritic cells (DC) are unique in that
they are the only cells capable of stimulating naive T cells, the major requirement of successful
vaccination. Lack of efficacy of DNA vaccines may be due to inefficient targeting of DC. One
strategy to improve DNA vaccines is to use cytokine gene adjuvants to recruit DC, such as GMCSF and IL-3.
The aim of the work carried out in this thesis was to investigate the effects of GM-CSF and IL-3
after gene-gun vaccination. Initial studies focused on the histological effects of GM-CSF and IL-3
in ovine skin. GM-CSF induced a linear increase in IL-lp transcripts from 1-24 hours concomitant
with pronounced neutrophilic infiltration and micro-abscess formation by 24 hours. Maximal GMCSF and IL-3 mRNA expression was evident at approximately 4 hours, whereas IL-ip and TNF-a
transcripts remained highly elevated up to 96 hours. Infiltration of DC, coordinately with a rise in
MHC class II DRa expression in perivascular regions, was observed 72-96 hours after GM-CSF
administration. Recruitment of eosinophils and neutrophils was evident 24-48 hours after
administration of IL-3, whereas infiltates of B cells and DC were observed at 24 and 72 hours,
Subsequent work characterised DC draining skin before and after GM-CSF administration by
means of cannulation of the pseudoafferent lymphatics. Two sub-populations of afferent lymph
DC (ALDC) were isolated based on differential expression of SIRPa as described in both cattle
and rats. Ovine SIRPa+ ALDC constitutively express high levels of IL-ip, IL-18 and IL-10
mRNA. Conversely, SIRPa ALDC do not express IL-10 but express high levels of IL-12p40.
SIRPa+ ALDC may contain Langerhans' cells since both ATPase and langerin were detected. Both
subpopulations express Toll like receptor 3 (TLR3) and TLR9, whereas SIRPa" ALDC uniquely
express TLR4 (+/- CD14). Gene-gun vaccination with pGM-CSF resulted in increased cytokine
transcripts in both ALDC subpopulations and an upregulation of surface MHC class Ila, CD lb,
CD40, CD86 and CD 11c. These studies have extended the knowledge of GM-CSF and IL-3 as
adjuvants and highlight the use of the cannulation model to further decipher the immune
mechanisms of adjuvants. This work has demonstrated that GM-CSF not only recruits DC into
skin but also activates DC, possibly as a result of increased expression of "danger signals"
including IL-1|3 and TNF-a.