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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.

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Summary of the last lecture: DC & the initiation of immune responses

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  1. 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 Summary of the last lecture:DC & the initiation of immune responses

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

  4. Antigens and antibodies Feedback and intracellular signaling Apoptosis and program cell death Neuro-endocrine-immune regulation Immune deviations and cytokines TH1 versus TH2 responses Immune regulation & homeostasis

  5. 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 TH1: nylon wool non-adherent, Ia- TH2: nylon wool adherent, Ia+ T helper cell functional heterogeneity

  6. CD4+ T cell subsets induced during infection or immunization with Leishmania Major in mice(Liew FY 1986) Cells Source Protection Ab IL-4 IFNg • Tr cured + + - + • Ti i.v. immunized + + - + • Ts progressive - + + - • Tsc s.c. immunized - + + - Refs.: Liew FY. Immunology Today 1989; 10:40-45 Liew FY. Parasitol. Today 1986; 2:264-270

  7. TH1: IL-2, IFN-g, GM-CSF, IL-3 TH2: BSF1 (IL-4), IL-3 … T helper clones distinct in cytokine production profiles(Mosmann TR, Coffman RL et al.,1986)

  8. TH0 TH3 TH cell subsets TGF-b TH1 TH2 inhibition IFN-g IL-4 Cell-mediated immunity Antibody production

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

  10. TH0 IL-12 IL-18 IL-4 TH1 TH2 inhibition IFN-g IL-4 Cell-mediated immunity Antibody production

  11. Factors important for TH1 differentiation IL-12 (MQ, DC) IL-18 (MQ, Kupffer cells, DC) IFN-g (T, NK, DC) Factors important for TH2 differentiation IL-4 (NK-T/mast cells? T, DC) IL-6 (T, MQ) IL-10 (T, MQ, DC) Factors driving TH cell differentiation NK-T: NK1.1+CD4+CD1+

  12. DC are heterogenic cell populations

  13. 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+) Types and source of DC

  14. DC lineage origins (mouse) Myeloid markers: CD11b, CD11c, 33D1 Lymphoid markers: CD8aa, DEC205, Key DC subsets Mouse: CD8aa- (myeloid) CD8aa+ (lymphoid) Human: DC1 (myeloid): e.g. PBMC-DC DC2 (lymphoid?): plasmacytoid DC (IL-3R++) Rat: OX62+OX41+ CD4+ OX62+OX41-CD4- DC subsets and lineage origins

  15. Mouse DC CD8aa- (myeloid): IL-10 CD8aa+ (lymphoid): IL-12, IFN-g Human DC DC1 (myeloid): IL-12, IL-18 DC2 (pDC, lymphoid?): IL-6, IL-10, IFN-a, IFN-b DC direct immune responses by releasing different cytokines

  16. Dendritic cell heterogeneity:immunogenicity vs tolerogenicity

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

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

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

  20. 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

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

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

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

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

  25. 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 Treg “Tolerogenic” “immunogenic”

  26. 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 DC-based immunotherapies

  27. Immature DC? DC “licensed to kill” Thymic medullary DC mediated negative selection CD8aa+ DC in mouse (Fas/FasL mediated killing) OX41-CD4- DC in rat (NKR-P1) DC associated tolerogenic molecules Cytokines: IL-10, TGF-b 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? Tolerogenic DC?

  28. Applications of Flow Cytometry in Basic Immunology • 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

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

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

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

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

  33. Fluorochromes & wave length

  34. Cell types of the immune system

  35. Human blood cells analyzed by their FSC and SSC properties Total blood RBC lysed SSC (granularity) FSC (size)

  36. Cell phenotypes & surface markers NK T B

  37. Cluster of Differentiation (CD) markers

  38. Immuno-staining of cell surface markers DirectIndirect

  39. Human blood leukocytes (back-gating analysis) CD14-FITC SSC CD45-PE FSC

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

  41. Markers of T cell differentiation

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

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

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

  45. T cell activation analyzedby CD25 (IL-2Ra) expression UnstimulatedCon A-stimulated Cell number CD25-PE SSC FSC

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