Dr Graham Ogg MRC Programme Leader, Oxford Consultant Dermatologist

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

2. 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

4. T cell recognises antigen presented by HLA class I/II TCR CD4/CD8 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. CD4+ T cell recognition of target cell leads to: • Cytokine production • Proliferation of T cell (clonal expansion) Th2 Th1 vs IL-4 production IgE class switching Eosinophil recruitment IFNg production CD8+ T cell help Macrophage activation IgG class switching

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: • Lysis of target cell • Cytokine production • Proliferation of T cell (clonal expansion)

19. ELISpot can be used to detect cytokine secreting cells positive control negative control pep4 pep12 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 Tissue Blood

22. 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

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. 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

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

27. Working model of disease Barrier Allergen Infection

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

29. Allergen-specific CD4+ T cells proliferate in vitro Ex vivo 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. 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

33. What about other forms of barrier compromise

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

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

36. Mapping Ves V5 epitopes

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

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

40. IFNg treated keratinocytes can engulf fluorescent latex particles

41. IFNg 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. 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

47. Depletion of IFNg 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-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