Dermatological Danger Dr Graham Ogg MRC Programme Leader

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Dermatological Danger Dr Graham Ogg MRC Programme Leader, Oxford Consultant Dermatologist

Dermatological Danger Dr Graham Ogg MRC Programme Leader, Oxford Consultant Dermatologist

Cutaneous inflammatory patterns

Cutaneous inflammatory patterns

Aim: To understand role of human cutaneous T cells in mechanisms of disease, treatment

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

T cell recognises antigen presented by HLA class I/II TCR CD 4/CD 8 HLA

T cell recognises antigen presented by HLA class I/II TCR CD 4/CD 8 HLA

HLA class II comprises 2 chains

HLA class II comprises 2 chains

T cell receptor and HLA class II

T cell receptor and HLA class II

HLA class II

HLA class II

How does the antigenic peptide get to HLA class II?

How does the antigenic peptide get to HLA class II?

Endosomes fuse with vesicles containing proteolytic enzymes

Endosomes fuse with vesicles containing proteolytic enzymes

These fuse with vesicles containing receptive HLA class II Invariant chain

These fuse with vesicles containing receptive HLA class II Invariant chain

Each HLA class II binds peptides carrying preferred motifs

Each HLA class II binds peptides carrying preferred motifs

CD 4+ T cell recognition of target cell leads to: 1. Cytokine production 2.

CD 4+ T cell recognition of target cell leads to: 1. Cytokine production 2. Proliferation of T cell (clonal expansion) Th 2 IL-4 production Ig. E class switching Eosinophil recruitment vs Th 1 IFNg production CD 8+ T cell help Macrophage activation Ig. G class switching

HLA class I

HLA class I

T cell receptor/HLA class I T cell receptor MHC class I

T cell receptor/HLA class I T cell receptor MHC class I

HLA Class I (T cell receptor view)

HLA Class I (T cell receptor view)

HLA class I antigen presentation proteasome

HLA class I antigen presentation proteasome

Some degraded peptides enter the endoplasmic reticulum

Some degraded peptides enter the endoplasmic reticulum

CD 8+ T cell recognition of target cell leads to: 1. Lysis of target

CD 8+ T cell recognition of target cell leads to: 1. Lysis of target cell 2. Cytokine production 3. Proliferation of T cell (clonal expansion)

ELISpot can be used to detect cytokine secreting cells negative control positive control pep

ELISpot can be used to detect cytokine secreting cells negative control positive control pep 4 pep 12 Bateman et al JACI 2006

HLA-peptide tetrameric complexes Ogg et al Science 1998 Champagne/Ogg et al Nature 2001 Seneviratne

HLA-peptide tetrameric complexes Ogg et al Science 1998 Champagne/Ogg et al Nature 2001 Seneviratne et al J Clin Invest 2002

HLA tetramers allow us to look at T cells that are specific for a

HLA tetramers allow us to look at T cells that are specific for a particular antigen Blood Tissue

Cells in the skin that might present antigen to T cells Keratinocytes Fibroblasts Melanocytes

Cells in the skin that might present antigen to T cells Keratinocytes Fibroblasts Melanocytes Others HLA class I Langerhans cells Dermal dendritic cells Keratinocytes (under inflamm conditions) HLA class II

Atopic dermatitis (eczema) • Cumulative prevalence up to 15 -20% • Onset usually by

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 Ig. E and/or skin test reactivity to common environmental allergens • FLG null mutations common

Atopic dermatitis – genetics and environment • Genome screens detected linkage to eg 3

Atopic dermatitis – genetics and environment • Genome screens detected linkage to eg 3 q 21, 17 q 25 and 20 p (similar to psoriatic susceptibility loci). Numerous candidate gene analyses eg Fce. RI, IL-4, IL 10, IL-13, SPINK 5, TLR 2. • Null mutations in FLG are commonly associated with atopic dermatitis Palmer et al Nature Genetics 2006

Filaggrin expression is variable and is inhibited by Th 2 cytokines Howell et al

Filaggrin expression is variable and is inhibited by Th 2 cytokines Howell et al JACI 2007

Severe atopic dermatitis is associated with common FLG null mutations in our cohort Cohort

Severe atopic dermatitis is associated with common FLG null mutations in our cohort Cohort 2282 del 4 hetero 2282 del 4 homo R 501 X hetero R 501 X homo Total >1 null mut 32 3 0 3 2 8

Working model of disease Barrier Allergen Infection

Working model of disease Barrier Allergen Infection

Individuals with atopic dermatitis have high frequencies of circulating allergen-specific Th 2 cells Non-atopics

Individuals with atopic dermatitis have high frequencies of circulating allergen-specific Th 2 cells Non-atopics Atopics Der p 1 peptides

Allergen-specific CD 4+ T cells proliferate in vitro Ex vivo Cultured ELISpot Ardern-Jones et

Allergen-specific CD 4+ T cells proliferate in vitro Ex vivo Cultured ELISpot Ardern-Jones et al 2007 PNAS

T cell epitope hunting

T cell epitope hunting

HLA-peptide tetrameric complexes Ogg et al Science 1998 Champagne/Ogg et al Nature 2001 Seneviratne

HLA-peptide tetrameric complexes Ogg et al Science 1998 Champagne/Ogg et al Nature 2001 Seneviratne et al J Clin Invest 2002

Individuals with atopic dermatitis have higher frequencies of circulating Der p 1 -specific CD

Individuals with atopic dermatitis have higher frequencies of circulating Der p 1 -specific CD 4+ T cells than non-atopics (short term culture) PATIENTS 1. 65% CONTROLS 5. 3% CD 4 AD 5 0. 02% AD 18 A 2. 34% AD 10 0. 54% 0. 01% AD 9 A 9. 9% AD 6 0. 29% 0. 44% AD 14 19. 13% AD 22 AD 25 0. 03% N 0. 02% J Tetramer

What about other forms of barrier compromise

What about other forms of barrier compromise

Wasp venom specific T cells responses Aslam et al Clin Exp Allergy 2006

Wasp venom specific T cells responses Aslam et al Clin Exp Allergy 2006

Dominant T cell antigens within wasp venom are coincident with main Ig. E binding

Dominant T cell antigens within wasp venom are coincident with main Ig. E binding proteins • Hyaluronidase • Antigen V • Phospholipase Aslam et al CEA 2006

Mapping Ves V 5 epitopes

Mapping Ves V 5 epitopes

Antigen-specific CD 4+ T cells infiltrate skin after antigen challenge PBMC Skin 0. 04%

Antigen-specific CD 4+ T cells infiltrate skin after antigen challenge PBMC Skin 0. 04% DRB 1*1501/IE 63 tetrameric complex 10%

IFNg increases class I, class II and ICAM-1 expression by keratinocytes CD 80 CD

IFNg increases class I, class II and ICAM-1 expression by keratinocytes CD 80 CD 86 CD 56 HLA-DR HLA-ABC Solid line = untreated Dashed line = overnight with IFN- ICAM-1

IFNg treated keratinocytes can engulf fluorescent latex particles

IFNg treated keratinocytes can engulf fluorescent latex particles

IFNg treated keratinocytes can present antigen to CD 4+ T cells using either peptide

IFNg treated keratinocytes can present antigen to CD 4+ T cells using either peptide or recombinant protein Black et al EJI 2007

Keratinocyte killing

Keratinocyte killing

sfu/40, 000 cells Increase in number of IL-4 -producing T cells using combined stimulation

sfu/40, 000 cells Increase in number of IL-4 -producing T cells using combined stimulation of Der p 1 -specific line with peptide and Staphylococcal enterotoxin B stimulus

Supernatant from SEB/PBMC enhances antigen presenting capacity of keratinocytes

Supernatant from SEB/PBMC enhances antigen presenting capacity of keratinocytes

IFNg within supernatant of SEB-treated PBMC enhances class II and ICAM-1 expression by keratinocytes

IFNg within supernatant of SEB-treated PBMC enhances class II and ICAM-1 expression by keratinocytes and enhances presentation to allergen-specific CD 4+ T cells Ardern-Jones et al

Depletion of IFNg from supernatant of SEB+PBMC diminishes ability of supernatant to promote keratinocyte

Depletion of IFNg 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 CD 4+ T cells

IL-4 depletion significantly reduces the production of cytokines by allergen-specific CD 4+ T cells Ardern-Jones et al

SEB-reactive T cells produce IFNg and IL-4 which enhances responsiveness of allergen-specific T cells

SEB-reactive T cells produce IFNg and IL-4 which enhances responsiveness of allergen-specific T cells SEB-reactive T cell Allergen-specific T cell IL-4 IFN-g SEB

Conclusions • FLG mutations associate with increased circulating airborne allergenspecific Th 2 cells. •

Conclusions • FLG mutations associate with increased circulating airborne allergenspecific Th 2 cells. • FLG mutations do not associate with circulating wasp venom-specific Th 2 cells. • These suggest that barrier factors plus Th 2 susceptibility important for allergic responses. • Keratinocytes can promote Th 2 responses • Antigen-specific CD 4+ 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 cofactor in determining Th 1/Th 2 response to a given antigen • MRC Experimental Medicine proof of concept clinical trial

Acknowledgements Louise Jones Neelika Malavige Antony Black UCL Milica Vukmanovic-Stejic Arne Akbar Tess Mc.

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