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

2. Cutaneous inflammatory patterns

3. 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

4. T cell recognises antigen presented by HLA class I/II CD4/CD8 TCR HLA

5. HLA class II comprises 2 chains

6. T cell receptor and HLA class II

7. HLA class II

8. How does the antigenic peptide get to HLA class II?

9. Endosomes fuse with vesicles containing proteolytic enzymes

10. These fuse with vesicles containing receptive HLA class II HLA class II Invariant chain

11. Each HLA class II binds peptides carrying preferred motifs

12. Th2 vs Th1 IFN  production CD8+ T cell help Macrophage activation IgG class switching IL-4 production IgE class switching Eosinophil recruitment CD4+ T cell recognition of target cell leads to: 1.Cytokine production 2.Proliferation of T cell (clonal expansion)

13. HLA class I

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

15. HLA Class I (T cell receptor view)

16. HLA class I antigen presentation proteasome

17. Some degraded peptides enter the endoplasmic reticulum

18. CD8+ T cell recognition of target cell leads to: 1.Lysis of target cell 2.Cytokine production 3.Proliferation of T cell (clonal expansion)

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

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

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

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

23. 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

24. Genome screens detected linkage to eg 3q21, 1q21, 17q25 and 20p (similar to psoriatic susceptibility loci). Numerous candidate gene analyses eg Fc  RI, 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

25. Filaggrin expression is variable and is inhibited by Th2 cytokines Howell et al JACI 2007

26. Severe atopic dermatitis is associated with common FLG null mutations in our cohort Cohort2282del4 hetero 2282del4 homo R501X hetero R501X homo Total >1 null mut 3230328

27. Working model of disease Barrier Allergen Infection

28. Individuals with atopic dermatitis have high frequencies of circulating allergen-specific Th2 cells Der p 1 peptides Non-atopics Atopics

29. Ex vivo Allergen-specific CD4+ T cells proliferate in vitro Cultured ELISpot Ardern-Jones et al 2007 PNAS

30. T cell epitope hunting

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

32. 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% Individuals with atopic dermatitis have higher frequencies of circulating Der p 1-specific CD4+ T cells than non-atopics (short term culture)

33. What about other forms of barrier compromise

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

35. Hyaluronidase Antigen V Phospholipase Dominant T cell antigens within wasp venom are co- incident with main IgE binding proteins Aslam et al CEA 2006

36. Mapping Ves V5 epitopes

38. Antigen-specific CD4+ T cells infiltrate skin after antigen challenge 10% 0.04% PBMCSkin DRB1*1501/IE63 tetrameric complex

39. CD80 HLA-DR CD86 CD56 HLA-ABCICAM-1 Solid line = untreated Dashed line = overnight with IFN-  IFN  increases class I, class II and ICAM-1 expression by keratinocytes

40. IFN  treated keratinocytes can engulf fluorescent latex particles

41. IFN  treated keratinocytes can present antigen to CD4+ T cells using either peptide or recombinant protein Black et al EJI 2007

42. Keratinocyte killing

44. 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

45. Supernatant from SEB/PBMC enhances antigen presenting capacity of keratinocytes

46. 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

47. Depletion of IFN  from supernatant of SEB+PBMC diminishes ability of supernatant to promote keratinocyte presentation of peptide

48. IL-4 depletion significantly reduces the production of cytokines by allergen-specific CD4+ T cells Ardern-Jones et al

49. SEB SEB-reactive T cell Allergen-specific T cell IL-4 IFN-  SEB-reactive T cells produce IFN  and IL-4 which enhances responsiveness of allergen-specific T cells

51. 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

52. 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-Stejic Arne Akbar UCL