Role of dendritic cells in response to cytokine gene adjuvants

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, respectively. 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.

Grantor dc:publisher

The University of Edinburgh

Year dc:date.issued

2006

Author dc:creator

• Matthews, Katie

Handle dc:identifier.uri

http://hdl.handle.net/1842/29874

OAI identifier oai:identifier

oai:era.ed.ac.uk:1842/29874