Dr Graham Ogg

1
Dermatological Danger
Dr Graham Ogg MRC Programme Leader,
Oxford Consultant Dermatologist
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Cutaneous inflammatory patterns
3
Aim To understand role of human cutaneous T
cells in mechanisms of disease, treatment and
vaccination
exogenous antigens eg atopic
HLA class II
HLA class I
endogenous antigens eg varicella zoster virus
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T cell recognises antigen presented by HLA class
I/II
TCR
CD4/CD8
HLA
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HLA class II comprises 2 chains
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T cell receptor and HLA class II
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HLA class II
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How does the antigenic peptide get to HLA class
II?
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Endosomes fuse with vesicles containing
proteolytic enzymes
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These fuse with vesicles containing receptive HLA
class II
HLA class II
Invariant chain
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Each HLA class II binds peptides carrying
preferred motifs
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• CD4 T cell recognition of target cell leads to
• Cytokine production
• Proliferation of T cell (clonal expansion)

Th2
vs
Th1
IFNg production CD8 T cell help Macrophage
activation IgG class switching
IL-4 production IgE class switching Eosinophil
recruitment
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HLA class I
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T cell receptor/HLA class I
T cell receptor
MHC class I
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HLA Class I (T cell receptor view)
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HLA class I antigen presentation
proteasome
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Some degraded peptides enter the endoplasmic
reticulum
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• CD8 T cell recognition of target cell leads to
• Lysis of target cell
• Cytokine production
• Proliferation of T cell (clonal expansion)

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ELISpot can be used to detect cytokine secreting
cells
positive control
negative control
pep 4
pep 12
Bateman et al JACI 2006
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HLA-peptide tetrameric complexes
Ogg et al Science 1998 Champagne/Ogg et al Nature
2001 Seneviratne et al J Clin Invest 2002
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HLA tetramers allow us to look at T cells that
are specific for a particular antigen
Tissue
Blood
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Cells in the skin that might present antigen to T
cells
Keratinocytes Fibroblasts Melanocytes Others
Langerhans cells Dermal dendritic
cells Keratinocytes (under inflamm conditions)
HLA class I
HLA class II
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Atopic dermatitis (eczema)

• Cumulative prevalence up to 15-20
• Onset usually by age 2-6 months
• 50-75 of children clear by age 10 years
• 50 have associated asthma and/or hayfever
• Staphylococcus aureus presence common (cf
  impetigo)
• 80 have IgE and/or skin test reactivity to
  common environmental allergens
• FLG null mutations common

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Atopic dermatitis genetics and environment

• Genome screens detected linkage to eg 3q21, 1q21,
  17q25 and 20p (similar to psoriatic
  susceptibility loci). Numerous candidate gene
  analyses eg FceRI, IL-4, IL-10, IL-13, SPINK5,
  TLR2.
• Null mutations in FLG are commonly associated
  with atopic dermatitis

Palmer et al Nature Genetics 2006
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Filaggrin expression is variable and is inhibited
by Th2 cytokines
Howell et al JACI 2007
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Severe atopic dermatitis is associated with
common FLG null mutations in our cohort
Cohort 2282del4 hetero 2282del4 homo R501X hetero R501X homo Total gt1 null mut
32 3 0 3 2 8
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Working model of disease
Barrier
Allergen
Infection
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Individuals with atopic dermatitis have high
frequencies of circulating allergen-specific Th2
cells
Non-atopics
Atopics
Der p 1 peptides
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Allergen-specific CD4 T cells proliferate in
vitro
Ex vivo
Cultured ELISpot
Ardern-Jones et al 2007 PNAS
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T cell epitope hunting
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HLA-peptide tetrameric complexes
Ogg et al Science 1998 Champagne/Ogg et al Nature
2001 Seneviratne et al J Clin Invest 2002
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Individuals with atopic dermatitis have higher
frequencies of circulating Der p 1-specific CD4
T cells than non-atopics (short term culture)
PATIENTS
CONTROLS
1.65
5.3
0.02
CD4
AD5
AD18
A
2.34
0.54
0.01
AD9
AD10
A
9.9
0.44
0.03
AD6
N
AD14
0.02
19.13
0.29
AD22
J
AD25
Tetramer
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What about other forms of barrier compromise
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Wasp venom specific T cells responses
Aslam et al Clin Exp Allergy 2006
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Dominant T cell antigens within wasp venom are
co-incident with main IgE binding proteins

• Hyaluronidase
• Antigen V
• Phospholipase

Aslam et al CEA 2006
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Mapping Ves V5 epitopes
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(No Transcript)
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Antigen-specific CD4 T cells infiltrate skin
after antigen challenge
PBMC
Skin
0.04
10
DRB11501/IE63 tetrameric complex
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IFNg increases class I, class II and ICAM-1
expression by keratinocytes
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IFNg treated keratinocytes can engulf fluorescent
latex particles
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IFNg treated keratinocytes can present antigen to
CD4 T cells using either peptide or recombinant
protein
Black et al EJI 2007
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Keratinocyte killing
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(No Transcript)
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Increase in number of IL-4-producing T cells
using combined stimulation of Der p 1-specific
line with peptide and Staphylococcal enterotoxin B
sfu/40,000 cells
stimulus
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Supernatant from SEB/PBMC enhances antigen
presenting capacity of keratinocytes
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IFNg within supernatant of SEB-treated PBMC
enhances class II and ICAM-1 expression by
keratinocytes and enhances presentation to
allergen-specific CD4 T cells
Ardern-Jones et al
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Depletion of IFNg from supernatant of SEBPBMC
diminishes ability of supernatant to promote
keratinocyte presentation of peptide
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IL-4 depletion significantly reduces the
production of cytokines by allergen-specific CD4
T cells
Ardern-Jones et al
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SEB-reactive T cells produce IFNg and IL-4 which
enhances responsiveness of allergen-specific T
cells
Allergen-specific T cell
SEB-reactive T cell
IL-4
IFN-g
SEB
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(No Transcript)
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• Conclusions
• FLG mutations associate with increased
  circulating airborne allergen-specific Th2 cells.
• FLG mutations do not associate with circulating
  wasp venom-specific Th2 cells.
• These suggest that barrier factors plus Th2
  susceptibility important for allergic responses.
• Keratinocytes can promote Th2 responses
• Antigen-specific CD4 T cells can infiltrate
  skin
• Combined presence of S.aureus and allergen
  enhances allergic inflammation.
• Handling of concurrent adjuvant is likely to be
  an important co-factor in determining Th1/Th2
  response to a given antigen
• MRC Experimental Medicine proof of concept
  clinical trial

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Acknowledgements
Louise Jones Neelika Malavige Antony Black Tess
McPherson Aamir Aslam Michael Ardern-Jones Laura
Crack Hsien Chan Carol Hlela Elizabeth Bateman
UCL
Milica Vukmanovic-Stejic Arne Akbar
Funding MRC NIHR