1. Major question: Recognition of microbes by dendritic cells (via TLRs) is essential to activate the adaptive response Viruses can be strictly intracellular and usually don’t infect DCs So, how do DCs present viral Ag??

2. Hypotheses:

3. Cells other than DCs (stromal cells) contribute to presentation of Ag TLRs of stromal cells recognize pathogen-associated molecular patterns (PAMPs)

4. Experimental system: Herpes simplex virus type 2 (HSV-2) Thymidine kinase (TK) mutant can infect but not replicate Infect vaginal epithelium of mice Examine response to mucosal infection Primarily measure activation of CD4 +  T H 1

5. Figure 1: IF-THEN??

6. Figure 1: IF TLR signalling is required for T H 1 response to HSV-2, THEN disrupting TLR signalling will block T H 1 response

7. Figure 1: Technique for measuring T H 1 response?

8. Figure 1: Technique for measuring T H 1 response: Infect mice with HSV-2 Isolate CD4 + cells from lymph nodes anti-CD4 on magnetic beads Stimulate in vitro with APCs or DCs ± HSV-2 Ag Assay cytokine production how?

9. Figure 1: Disruption of TLR signalling: MyD88 is an adapter protein in the TLR signal transduction pathway Knockout mice (MyD88 –/–) lack this protein (why is this better than knocking out a TLR?)

10. Figure 1: Activated cells in WT make IFN- , not IL-4 or 10 Activated cells in KO make IL-4 and 10, not IFN-  APCsDCs Significance? Controls?

11. Figure 1: Activated cells in WT make IFN- , not IL-4 or 10 Activated cells in KO make IL-4 and 10, not IFN-  TLR signalling is needed for T H 1 response APCsDCs

12. Figure 2: IF-THEN??

13. Figure 2: IF TLR signalling is required for DC recruitment to mucosa, THEN disrupting TLR signalling will block recruitment

14. Figure 2: Technique for measuring recruitment to mucosa?

15. Figure 2: Technique for measuring recruitment to mucosa: Immunofluorescence staining (in situ) anti-CD11c Ab: specific DC marker

16. Figure 2: Similar recruitment of DCs to infected epithelium KOWT blue = nuclei red = DCs green = virus

17. Figure 3: IF-THEN??

18. Figure 3: IF TLR signalling is required for DC migration to lymph nodes or maturation, THEN disrupting TLR signalling will block migration or maturation

19. Figure 3: Technique for measuring migration to lymph nodes and maturation?

20. Figure 3: Technique for measuring migration to lymph nodes and maturation: Fluorescence- activated cell sorting (FACS)

21. Figure 3: Technique for measuring migration to lymph nodes and maturation: Fluorescence- activated cell sorting (FACS)

22. Figure 3: Technique for measuring migration to lymph nodes and maturation: Fluorescence-activated cell sorting (FACS) DCs are: CDC11c + / B220 – / CD8 – Mature DCs are also CD86 + Isolate CD11c + cells Sort with fluorescent anti-CD8 and anti-CD86

23. Figure 3: TLR signalling does not change numbers of CD4 or DC cells in lymph nodes

24. Figure 3: TLR signalling does not change numbers of CD4 or DC cells in lymph nodes TLR signalling does not change number or maturity of DCs WT KO MockHSV mature DCs

25. Figure 3: TLR signalling does not change numbers of CD4 or DC cells in lymph nodes TLR signalling does not change number or maturity of DCs TLR signalling is not needed for migration or maturity WT KO MockHSV mature DCs

26. Figure 4: Possible explanations for Fig. 1 results: T H 1 activation requires TLR signalling T H 1 activation requires IL-1 or IL-18 (also dependent on MyD88) T H 1 activation requires IL-12 T H 1 activation requires IFN- 

27. Figure 4: IF-THEN??

28. Figure 4: IF MyD88 KO mice lack T H 1 response due to lack of IL-1 (or IL-18), IL-12 or IFN- , THEN lack of one of these products would have the same effect as the MyD88 KO

29. Figure 4: Technique for measuring effects of cytokines on T H 1 activation?

30. Figure 4: Technique for measuring effects of cytokines on T H 1 activation: Additional KO mice: caspase-1 (IL-1  -converting enzyme) KO mice: lack functional IL-1 IL-12 p40 KO mice: lack functional IL-12 IFN-  R KO mice: DCs can’t respond to IFN-  Same cytokine assay as Fig. 1

31. Figure 4: Lack of IL-1 does not affect T H 1 activation Lack of IL-12 does not affect T H 1 activation Lack of DC response to IFN-  does not affect T H 1 activation APCsDCs

32. Figure 4: Lack of IL-1 does not affect T H 1 activation Lack of IL-12 does not affect T H 1 activation Lack of DC response to IFN-  does not affect T H 1 activation Activation appears to specifically require TLR signal APCsDCs

33. Figure 5A: IF-THEN??

34. Figure 5A: IF epithelial cells are involved in TLR signalling, THEN these cells must express TLR genes

35. Figure 5A: Technique for measuring expression of TLR genes?

36. Figure 5A: Technique for measuring expression of TLR genes: RT-PCR Primer pair specific for each TLR gene Isolate mRNA Reverse transcriptase  DNA PCR to amplify: band indicates mRNA was present

37. Figure 5A: Epithelial cells express all tested TLR genes

38. Figure 5B-E: IF-THEN??

39. Figure 5B-E: IF TLR signalling by epithelial cells is needed for T H 1 activation THEN MyD88 KO in epithelial cells will block T H 1 activation even if WT DCs are present

40. Figure 5B-E: Technique for making mice with genetically different epithelial and DC cells???

41. Figure 5B-E: Technique for making mice with genetically different epithelial and DC cells: Bone marrow (BM) chimera: lethal irradiation of mouse to kill bone marrow cells “bone marrow transplant” from a different strain epithelial cells have original genotype; DCs have donor genotype WTMyD88 KO BM cells MyD88 (-/-) DCs MyD88 + epithelium KO BM  WT

42. Figure 5B-E: Technique for making mice with genetically different epithelial and DC cells: Bone marrow (BM) chimera Same cytokine assay for CD4 cell activation

43. Figure 5B-E: MyD88 required in DCs for activation MyD88 (-/-) DCs MyD88 + epithelium APCsDCs

44. Figure 5B-E: MyD88 required in epithelial cells for activation MyD88 (-/-) epithelium MyD88 + DCs APCsDCs

45. Figure 5B-E: MyD88 required in DCs for activation MyD88 required in epithelial cells for activation Both DCs and stromal cells participate in Ag presentation and require TLR signalling APCsDCs

46. Figure 6: IF-THEN??

47. Figure 6: IF signalling by a specific TLR is required for T H 1 activation, THEN KO of that TLR in stromal cells and/or DCs will block T H 1 activation

48. Figure 6: Technique for testing TLR KO in DC and stromal cells?

49. Figure 6: Technique for testing TLR KO in DC and stromal cells? Chimera with WT DCs and TLR KO stromal cells or vice- versa Same cytokine assay

50. Figure 6: TLR9 not required for activation Not shown: TLR2, TLR3, TLR4 also not required

51. Conclusions: Stromal cells and DCs are involved in presentation of intracellular virus Ag to T H cells Requires TLR signalling by both stromal cells and DCs