Dr Graham Ogg MRC Programme Leader, Oxford Consultant Dermatologist Dermatological Danger.
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Transcript of Dr Graham Ogg MRC Programme Leader, Oxford Consultant Dermatologist Dermatological Danger.
Dr Graham OggMRC Programme Leader, Oxford
Consultant Dermatologist
Dermatological Danger
Cutaneous inflammatory patterns
exogenous antigens eg atopic
endogenous antigens eg varicella zoster virus
Aim: To understand role of human cutaneous T cells in mechanisms of disease, treatment and vaccination
HLA class I HLA class II
T cell recognises antigen presented by HLA class I/II
CD4/CD8TCR
HLA
HLA class II comprises 2 chains
T cell receptor and HLA class II
HLA class II
How does the antigenic peptide get to HLA class II?
Endosomes fuse with vesicles containing proteolytic enzymes
These fuse with vesicles containing receptive HLA class II
HLA class II
Invariant chain
Each HLA class II binds peptides carrying preferred motifs
Th2 vs Th1
IFN productionCD8+ T cell helpMacrophage activationIgG class switching
IL-4 productionIgE class switchingEosinophil recruitment
CD4+ T cell recognition of target cell leads to:
1. Cytokine production2. Proliferation of T cell (clonal expansion)
HLA class I
T cell receptor/HLA class I
T cell receptor
MHC class I
HLA Class I (T cell receptor view)
HLA class I antigen presentation
proteasome
Some degraded peptides enter the endoplasmic reticulum
CD8+ T cell recognition of target cell leads to:
1. Lysis of target cell2. Cytokine production3. Proliferation of T cell (clonal expansion)
pep 4negativecontrol
pep 12
ELISpot can be used to detect cytokine secreting cells
positivecontrol
Bateman et al JACI 2006
HLA-peptide tetrameric complexes
Ogg et al Science 1998Champagne/Ogg et al Nature 2001Seneviratne et al J Clin Invest 2002
HLA tetramers allow us to look at T cells that are specific for a particular antigen
Blood Tissue
HLA class I HLA class II
KeratinocytesFibroblastsMelanocytesOthers
Langerhans cellsDermal dendritic cellsKeratinocytes (under inflamm conditions)
Cells in the skin that might present antigen to T cells
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
•Genome screens detected linkage to eg 3q21, 1q21, 17q25 and 20p (similar to psoriatic susceptibility loci). Numerous candidate gene analyses eg FcRI, IL-4, IL-10, IL-13, SPINK5, TLR2.
•Null mutations in FLG are commonly associated with atopic dermatitis
Atopic dermatitis – genetics and environment
Palmer et al Nature Genetics 2006
Filaggrin expression is variable and is inhibited by Th2 cytokines
Howell et al JACI 2007
Severe atopic dermatitis is associated with common FLG null mutations in our cohort
Cohort 2282del4
hetero
2282del4
homo
R501X
hetero
R501X
homo
Total >1 null mut
32 3 0 3 2 8
Working model of disease
Barrier
Allergen Infection
Individuals with atopic dermatitis have high frequencies of circulating allergen-specific Th2 cells
Der p 1 peptides
Non-atopics
Atopics
Ex vivo
Allergen-specific CD4+ T cells proliferate in vitro
Cultured ELISpot
Ardern-Jones et al 2007 PNAS
T cell epitope hunting
HLA-peptide tetrameric complexes
Ogg et al Science 1998Champagne/Ogg et al Nature 2001Seneviratne et al J Clin Invest 2002
1.65%
2.34%
0.29%
5.3% 0.02%
0.01%0.54%
0.44%
PATIENTS CONTROLS
CD4
Tetramer
AD5
AD6
AD10
AD22
AD25
AD18
AD9
AD14
A
0.03%
N
J
A
0.02%19.13%
9.9%
AD controls0.001
0.01
0.1
1
10
100 P<0.05
Perc
enta
ge C
D3+
and
Tetr
amer
+
Individuals with atopic dermatitis have higher frequencies of circulating Der p 1-specific CD4+ T cells than non-atopics
(short term culture)
What about other forms of barrier compromise
Wasp venom specific T cells responses
Aslam et al Clin Exp Allergy 2006
•Hyaluronidase
•Antigen V
•Phospholipase
Dominant T cell antigens within wasp venom are co-incident with main IgE binding proteins
Aslam et al CEA 2006
Mapping Ves V5 epitopes
Antigen-specific CD4+ T cells infiltrate skin after antigen challenge
10%0.04%
PBMC Skin
DRB1*1501/IE63 tetrameric complex
CD80
HLA-DR
CD86
CD56
HLA-ABC ICAM-1
Solid line = untreatedDashed line = overnight with IFN-
IFN increases class I, class II and ICAM-1 expression by keratinocytes
IFN treated keratinocytes can engulf fluorescent latex particles
IFN treated keratinocytes can present antigen to CD4+ T cells using either peptide or recombinant protein
Black et al EJI 2007
Keratinocyte killingCell count 040506
Nil 1:1 10:1 40:10
250
500
750
1000Clone 1Clone 8Clone 10Clone 14
Nu
mb
er /
10x
fie
ld
Increase in number of IL-4-producing T cells using combined stimulation of Der p 1-specific line with
peptide and Staphylococcal enterotoxin Bsf
u/40
,000
cel
ls
stimulus
Supernatant from SEB/PBMC enhances antigen presenting capacity of keratinocytes
IFN 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
Depletion of IFN from supernatant of SEB+PBMC diminishes ability of supernatant to promote keratinocyte presentation of
peptide
IL-4 depletion significantly reduces the production of cytokines by allergen-specific CD4+ T cells
Ardern-Jones et al
SEB
SEB-reactiveT cell
Allergen-specificT cell
IL-4
IFN-
SEB-reactive T cells produce IFN and IL-4 which enhances responsiveness of allergen-specific T cells
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
Acknowledgements
Louise Jones
Neelika Malavige
Antony Black
Tess McPherson
Aamir Aslam
Michael Ardern-Jones
Laura Crack
Hsien Chan
Carol Hlela
Elizabeth Bateman
Funding
MRC
NIHR
Milica Vukmanovic-StejicArne Akbar
UCL