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