1. Summary of the last lecture: DC & the initiation of immune responses Lymphocyte activation – the requirements – Signal 1 – Signal 2 Models of T-B, APC-T-B cell cooperation DC – the initiator of immunity – Activator of naïve T cells – The special Ag processing & presenting machine The “‘Self’ vs ‘Non-self’” and “Danger” models

2. Inflammation & “ danger signals ” – Immune System Turned on by “Danger” (P Matzinger)  Clinical signs of inflammation:  erythema, swelling, heat & pain  Mediators:  Cytokines: IL-1, IL-6, TNF  (endo-pyrogen) & chemokines  Acute phase proteins, PG, histamine  Bacterial products: LPS  endogenous & exogenous “danger signals”  Immunological effects:  vasodialation, vascular permeability  cell adhesion, chemotaxis – cell mobilization & immobilization  DC activation

3. Dendritic cells & immune regulation Immune regulation & deviations DC heterogeneity DC & immune regulation Applications of flow cytometry in basic immunology Student presentation & essay topic

4. Immune regulation & homeostasis Antigens and antibodies Feedback and intracellular signaling Apoptosis and program cell death Neuro-endocrine-immune regulation Immune deviations and cytokines – T H 1 versus T H 2 responses

6. T helper cell functional heterogeneity Liew FY & Parish CR. (1972,1974): Distinct T helper functions for: – Ab response to hapten-carriers – DTH responses to the carrier protein Tada T et al. (1978): T helper types for Ab responses to different hepten-carrier conjugates – T H 1: nylon wool non-adherent, Ia - – T H 2: nylon wool adherent, Ia +

7. CD4 + T cell subsets induced during infection or immunization with Leishmania Major in mice (Liew FY 1986) CellsSourceProtectionAbIL-4IFN  Trcured++-+ Tii.v. immunized++-+ Tsprogressive-++- Tscs.c. immunized-++- Refs.: Liew FY. Immunology Today 1989; 10:40-45 Liew FY. Parasitol. Today 1986; 2:264-270

8. T helper clones distinct in cytokine production profiles (Mosmann TR, Coffman RL et al.,1986) T H 1: IL-2, IFN- , GM-CSF, IL-3 T H 2: BSF1 (IL-4), IL-3 …

9. T H cell subsets TH0TH0 TH2TH2TH1TH1 IFN-  IL-4 Cell-mediated immunity Antibody production inhibition TH3TH3 TGF- 

10. Type of immune effector mechanism induced may determine the outcome of an infection

13. TH0TH0 TH2TH2TH1TH1 IFN-  IL-4 Cell-mediated immunity Antibody production inhibition IL-12 IL-18 IL-4

14. Factors driving T H cell differentiation Factors important for T H 1 differentiation – IL-12 (MQ, DC ) –IL-18 (MQ, Kupffer cells, DC ) – IFN-  (T, NK, DC ) Factors important for T H 2 differentiation –IL-4 (NK-T/mast cells? T, DC ) –IL-6 (T, MQ) – IL-10 (T, MQ, DC ) NK-T: NK1.1 + CD4 + CD1 +

15. DC are heterogenic cell populations

16. Types and source of DC Bone marrow-derived (BM-DC) – Can be generated in the presence of GM-CSF and IL-4 Peripheral mononulear cell-derived DC (PBMC-DC) – Can be generated in the presence of GM-CSF and IL-4 DC isolated from tissues and organs –Thymic DC –Langhan’s cells –Spleen, lymph node DC –Intestinal, Peyer’s patch, liver, lung DC –Migrating DC in the lymph Plasmacytoid DC –CD11C -, IL-3R +, B220 +, GR-1 + )

17. DC subsets and lineage origins DC lineage origins (mouse) – Myeloid markers: CD11b, CD11c, 33D1 – Lymphoid markers: CD8 , DEC205, Key DC subsets –Mouse: CD8  - (myeloid) CD8  + (lymphoid) –Human: DC1 (myeloid): e.g. PBMC-DC DC2 (lymphoid?): plasmacytoid DC (IL-3R ++ ) –Rat: OX62 + OX41 + CD4 + OX62 + OX41 - CD4 -

18. DC direct immune responses by releasing different cytokines Mouse DC –CD8  - (myeloid): IL-10 –CD8  + (lymphoid): IL-12, IFN-  Human DC –DC1 (myeloid): IL-12, IL-18 –DC2 (pDC, lymphoid?): IL-6, IL-10, IFN- , IFN- 

19. Dendritic cell heterogeneity: immunogenicity vs tolerogenicity

20. The “micro-anatomical model” (by B. Fazekas de St Groth, Imm.Today 1998; 19:448-54) B B B T

22. + DC GC (B + FDC) F T: T cell area B: B cell area F: B cell follicle GC: germinal centre Missions & fate of DC migrating to the secondary lymphoid organs

23. - A model for generating ‘authentic’ DC migrating in lymph (G. Gordon MacPherson, Oxford)

24. Two DC subsets in rat OX41 + CD4 + DC Smaller with short/fine processes Contain low/no NSE reactivity (Liu L.M. et. al. J. Immunol. 1998) OX41 - CD4 - DC larger with long/big processes Contain strong NSE reactivity

25. Presentation of KLH to sensitised T cells by DC subpopulations

26. Intestinal DC populations OX41 + DCOX41 - DC Strong APCWeak APC Not present in T cell areasContinuously transport under steady state self-Ag derived from apoptotic IEC to T cell areas under steady ‘non-danger’ state Express high levels of MHC Class II Express CD80 & CD86 (B7.1, B7.2) Acquire enteric antigens

27. Features that change during DC maturation process (Banchereau J & Steinmain RM, Nature 1998) “Ag uptake mode”“Ag presenting mode” (danger signals)

28. Distribution of MHC Class II molecules on DC (Turley S. & Mellman I.) Ref: Banchereau J & Steinman RM, Nature 1998 Early Intermediate Late Lamp-2 MHC class II

29. DC maturity & immuno-adjuvantivity Immature DC Mainly intracellular MHC Lack co-stimulatory molecules Mature DC High surface MHC High B7 (CD80, CD86) … T T T T T T T T “Tolerogenic”“immunogenic” Treg

30. DC-based immunotherapies DC-based tumor vaccines for cancer treatment –Immunodeficiencies and tumors –Graft-versus-leukemia (GVL) responses –Anergized TAA-specific T cell clones –DC immune adjuvanticity – DC subsets and maturity Immunotherapeutic potential of tolerogenic DC –Tolerance induction in autoimmune diseases –Tolerance induction in transplantation

31. Tolerogenic DC? Immature DC? DC “licensed to kill” –Thymic medullary DC mediated negative selection –CD8  + DC in mouse (Fas/FasL mediated killing) –OX41 - CD4 - DC in rat (NKR-P1) DC associated tolerogenic molecules –Cytokines: IL-10, TGF-  –IDO (Indoleamine 2,3-dioxygenase) DC and the generation of T regulatory cells – DC confers tolerogenic memory and specificity through the induction of T regulatory cells?

33. –Basic parameters –Applications Cell phenotyping Measurement of cell activation Visualization of cell division Detection of intracellular molecues (e.g. cytokine) Analysis of specific ligand binding Analysis of intracellular calcium concentration Analysis of DNA content Cell sorting Applications of Flow Cytometry in Basic Immunology

34. FSC (forward scatter) - Size SSC (side scatter) - Granularity FL (flurochrome) – FL-1, FL-2, FL-3 etc. Flow cytometry - the basic parameters

35. (1) FSC – the size Forward (parallel) lights

36. (2) SSC – the granularity Side (angled) lights

37. (3) Fluorescent-labeled markers Laser light Band Filters mirrors FL-1 (green) FL-3 (ultra red) FL-2 (red) Fluorescent detectors

38. Fluorochromes & wave length

39. Cell types of the immune system

40. Human blood cells analyzed by their FSC and SSC properties FSC (size) SSC (granularity) RBC lysedTotal blood

41. Cell phenotypes & surface markers TBTB NK

42. Cluster of Differentiation (CD) markers

43. Immuno-staining of cell surface markers DirectIndirect

44. Human blood leukocytes (back-gating analysis) CD14-FITC CD45-PEFSC SSC

45. Human PBL phenotyping Isotype-FITC Isotype-PE SSC Anti-CD3-FITC FSC Anti-CD19-PE Anti-CD8-PE Anti-CD4-PE Anti-CD56/16-PE Anti-CD3-FITC

46. Markers of T cell differentiation

47. CD4 and CD8 expression on thymocytes, splenocytes and PBL Thymocytes Splenocytes PBL CD8-FITC CD4-Cyt5

48. FP090502.015-021 GM GM/IL-4 MHC-Class II-PE Immature DC Mature DC +LPS DC maturity & MHC class II expression

49. Cell activation and proliferation analyzed by the FSC and SSC properties FSC (size) SSC (granularity) Unstimulated Con A-stimulated Dot plots Density plots

50. T cell activation analyzed by CD25 (IL-2R  ) expression CD25-PE Cell number Unstimulated Con A-stimulated SSC FSC

51. Th1 & Th2 Phenotypes Determined By Cytokine Expression Profile

52. CFSE: Carboxyfluorescein diacetate Succinimidyl Ester Analysis of cell division by CFSE labeling Cell incubation with CFSE (serum free, 37 0 C, 10mins. & wash) CFSE crosses cell membrane & attaches to cytoplasmic proteins Removal of carboxyl groups of CFSE by intracellular esterase Fluorescein Analyed by FC Excitation wave length: 488nm Emitting wave length: 519nm 1½¼...1½¼...

53. Visualizing lymphocyte division CFSE intensity

54. Applications of flow cytometry in basic immunology Generation and characterization of DC Assays for T cell activation –Cell proliferation – Cell division – Cell activation marker Laboratory demonstration - DC and T cell activation

55. Student Presentation & Essay Topic Topic: Regulatory T cells  Autoimmunity  Tumor immunity  Transplantation Tasks: 1.Class presentation  December 7, 2005 (5-9pm)  3 per group  20 – 25 minutes for presentation (per group)  10 minutes for questions & discussions  Optional for MMedSc students 2.Write an essay on the topic  All students  1000 -1500 words  Essay submission: by January 18, 2006 Contact:: Dr. FP Huang (Tel:2855 4864; e-mail: fphuang@hkucc.hku.hk)