1. Antigen presenting cells Antigen presenting cells - (1) dendritic cells (2) macrophages (3) B cells

2. Morphology of Dendritic Cells

3. Paradigm: Immunity is the result of co-evolution of microorganisms and the immune system Gram-Gram+Fungi INFECTIOUS NON SELF Virus Parasites Dendritic cells (DC): the sentinels of the immune system

4. DC orchestrate both innate and adaptive immunity Phagocytosing microorganisms Participating to inflammatory responses Activating appropriate T cell responses

5. HSC Myeloid- Lymphoid Precursor Myeloid precursor Lymphoid precursor Common DC precursor Monocytes CD8  - and CD8  + DC Langerhans cells Plasmacytoid cells ? MOUSE DCs HUMAN DCs HSC Myeloid- Lymphoid Precursor Myeloid precursor Common DC precursor Lymphoid precursor Monocytes CD11b+CD33+ DC Langerhans cells Plasmacytoid cells

6. DCs progenitors are generated in the bone marrow. They give rise to circulating DCs precursors. Circulating DCs precursors enter nonlymphoid tissues as immature DCs. DC are scattered throughout all non lymphoid tissues where they reside in a resting, (so-called immature) state. In the absence of ongoing inflammatory and immune responses, they constantly migrate at low rate to draining lymph nodes. DCs precursors Immature DCs DCs progenitors Mature DCs In inflammatory conditions, immature DC migrate to draining lymph nodes where after maturation (mature DC), they prime the rare circulating naïve antigen-specific lymphocytes.

7. There are no lineage-specific surface markers that are expressed on all DC. Moreover, DC are a heterogenous cell population. However, it has been clearly demonstrated that at least 3 distinct types of DC can be generated. Mice: myeloid DC CD11b+CD11c+, myeloid DC CD11b+ CD11c+/-, CD4+, lymphoid DC CD11b- CD8  + The existence of different subsets of DC has lead to possibility that they can perform unique functions.

8. Dendritic Cell Subpopulations

9. Plasmacytoid Dendritic Cells  A specialized subpopulation of DC.  Resemble plasma cells in morphology – also express some B cell surface markers (e.g. CD45R [B220]).  In response to some viral infections, pDC synthesize high levels of type I IFNs – cytokines have direct antiviral effects and activate NK cells.  Plasmacytoid DC also are a significant source of IL-12 during the early response – enhance IFN-  production by NK cells, and, subsequently, CD4 and CD8 T cells.

10. Process of Dendritic Cell Migration to Lymph Nodes

11. Growth factor-dependent DC Immature DCMature DCApoptotic DC GM-CSF or GM-CSF IL-4 conditioned medium bone marrow or peripheral blood Maturation stimulus

12. conventional phagocytosis macro pinocytosis triggered membrane- ruffling forced endocytosis coiling phagocytosis phagosomesmacro- pinosomes spacious vacuoles phagosomesphagosomes/ cytosol/replicative vacuoles Binding of bacteria to the cell surface Actin polymerizationpseudopod extension around the cell surface Engulpment Actin depolymerization Phagosome maturation transport event Phagocytosis by DC

14. The innate repertoire: the specificity of DC recognition is mediated by an extended family of receptors binding is mediated by an extended family of receptors binding a variety of ligands Immature DC Fc  R  Fc  R  Fc  R  CR3 (Mac-1) CD11c ScavengerR TLR 4: LPS/ Gram- DEC205 CD14 Collectins PAMP‘s PAMP‘s : Pathogen-Associated Molecular Patterns TLR 2: LTA/ Gram+ Gram - bacteria Gram + bacteria Fungi viruses Poli I:C CpG LPS/LTA TLR 9: CpG TLR 5: flagellinR conserved structures produced only by microorganisms but not by the host MARCO R

15. TLR2/1TLR2/6TLR3TLR4TLR7TLR8TLR10TLR9TLR5 zymosan, peptidoglycan & diacylated bacterial lipopeptides E. coli LPS, hsp60, RSV F protein bacterial glycolipids & triacylated bacterial lipopeptides ?? Flagellin CpG DNA, chromatin dsRNA Imiquimod Toll-like receptors CD14

16. Toll-like receptor signaling pathways

17. Toll-like receptor signaling pathways

18. Maturation process Gram-Gram+Fungi INFECTIOUS NON-SELF PAMP’s cytokines Virus NK Mature DC T cell activation CD4 CD8 Immature DC Innate responses Adaptive responses LPS, LTA, CpG, PGN Regulated expression of key molecules for the Initiation of adaptive immune responses cytokines

19. MATURATION PROCESS Migratory activity Cytoskeleton rearrangements MHC and costimulatory molecules upregulation

20. Migration Maturing DCs migrate into the T cell area of lymphoid organs. Immature Dcs express a variety of chemokine receptors (CCR1, CCR5, CXCR1 and CCR6) that participate in their recruitment to inflammed tissue and/or to allow their residency into non-lymphoid tissue. Exemple: Imm. Dcs express CCR6 the receptor for MIP-3a that is constitutively express in liver and lungs. Upon maturation, there is a downregulation of receptors for chemokines produced at the site of inflammation and upregulation of CCR7. SLC is a ligand for CCR7 and is expressed at high levels by HEVs in LNs and by stromal cells in T cell areas of many secondary lymphoid organs. ELC (other ligand of CCR7) made in T cell areas of lymphoid tissue. SLC (6Ckine) and ELC (MIP-3b) act together to direct DC migration to T cell areas Of lymphoid tissue and to promote encounter with T cells. Adhesion molecules Banchereau J. et al. Immunobiology of dendritic cells Ann. Rev. Immunol. 18:767-811, 2000.

21. Expression of Chemokine Receptors on Dendritic Cells ReceptorLigand Immature DC CCR1 CCR2 CCR4 CCR5 CCR6 CXCR1 CXCR4 Mature DC CCR7 MIP-1 , RANTES, MCP-3, MIP-5 MCPs TARC, MDC MIP-1 , MIP-1 , RANTES MIP-3  IL-8 SDF-1 MIP-3 , SLC (6Ckine) Abbreviations: DC, dendritic cell; MIP, macrophage inflammatory protein; RANTES, regulated on activation, normal T cell expressed and secreted; MCP, monocyte chemoattractant protein; TARC, thymus and activation-regulated chemokine; MDC, monophage derived chemokine; SDF, stromal derived factor; IL, interleukin; SLC, secondary lymphoid-tissue chemokine.

22. ControlLPS 0.5 hLPS 1h CLPS 2 hLPS 3 hLPS 24h 20 sec [Ca 2+ ] : nM Functional downregulation of chemokine receptor expression during DC maturation FURA-2 loaded D1 cells stimulated with MIP-1  L32 GAPDH CCR1 030’1 h2 h3 h24 h Time LPS -+++++ RNAse protection assay

23. Developmental Stages of Dendritic Cells Developmental stages of dendritic cells (DCs) in vivo. The generation of DC precursors in the bone marrow, the recruitment of immature DCs in peripheral tissues, and the migration of DCs into the lymphoid organs are illustrated. The maturation of DCs into potent antigen-presenting cells in case of infection or inflammation and their migration have been amply documented (right), but there is also evidence that in the "steady state“ – that is, in the absence of a "danger signal“ – these immature DCs may migrate into the lymphoid organs while remaining at the immature stage (left). The phenotype of the DC migrating in baseline conditions is still unclear. The movement of maturing DCs and the "constitutive" migration of immature DCs have been shown to depend on chemokine gradients.

24. MATURATION PROCESS Migratory activity Cytoskeleton rearrangements MHC and costimulatory molecules upregulation

25. Spleen-derived DCs

26. control cells MARCO- positive cells

27. Overview of Dendritic Cell Maturation

28. MATURATION PROCESS Migratory activity Cytoskeleton rearrangements MHC and costimulatory molecule upregulation

29. Molecules Involved in T Cell – DC Interactions

30. Dendritic Cells in Association with T Cells in vivo.

31. Innate immunity regulates the expression of key molecules for the initiation of the adaptive immune response B71/2CD40MHC II MHC l S. thyphimurium E. coli l. monocytogenes M.smegmatis BCG S. aureus S. pyogenes S. pneumococus S. gordonii Lactococco Lactobacillo Immature Mature MHC class II MHC class I B7.1 B7.2CD402.4G2

33. Peptides bind to MHC molecules through structurally related anchor residues

34. Peptides that bind MHC class II molecules are variable in lenght

35. The expression of MHC molecules differs between tissues

36. Golgi Proteasome + PA28 Peptides +/- Ubiquitin Endogenous proteins Exogenous proteins Antigen HLA class I synthesis  2m ERp57 Calreticulin Tapasin Endoplasmic reticulum Calnexin TAP MHC class I loading

37. M. Rescigno PNAS 1998

38. Cross-Presentation of Antigen by Dendritic Cells  Cross-presentation is the ability of dendritic cells to deliver exogenous antigens to the class I MHC processing and presentation pathway for the activation of CD8 T cells.  The process is rapid, occurring within 3 to 4 hours after antigen uptake.  The process requires a functional endocytic pathway –also requires a functional TAP complex, proteosome function and normal transport from the ER-Golgi to the cell surface.  The process is inhibited if the phagocytic and macropinocytic activities of the DC are inhibited.  Suggests that the DC degrades exogenous antigen in a lysosomal compartment, and then releases relatively long- length peptides into the cytoplasm, where they are ubiquinated, further cleaved by the proteosome, and then delivered to the ER by TAP.

39. Soon after or during formation, phagosomes fuse with the ER. After antigen export to the cytosol and degradation by the proteasome, peptides are translocated by TAP into the lumen of the same phagosomes, before loading on phagosomal MHC class I molecule. Therefore, cross-presentation in dendritic cells occurs in a specialized, self-sufficient, ER- phagosome mix compartment. Peptides derived from phagocytosed antigens can be presented to CD8+ T cells on MHC class I. Phenomenon called CROSS-PRESENTATION

40. Dendritic Cells Deliver Exogenous Antigens to the Class I MHC Pathway

41. The Class II MHC Antigen Processing and Presentation Pathway

42. Function of HLA-DM (H-2M in Mice)

43. HLA-DM is Physically Located In the MIIC Vesicle

44. M. Rescigno PNAS 1998

45. Phenotypic Changes Associated with Antigen Presentation

46. Immature DC are less efficient in MLR when compared to mature DC 20 40 60 80 100 120 100 10 10,1 cpm x 10 -3 0 APC (x 10 -3 ) MLR mature immature

47. . mDC iDC No DCs CD4 CFSE 48 h 72 h FSC Allogeneic CD4 T cell proliferation

48. iDC mDC CD8 48 h 72 h No DCs FSC CFSE Allogeneic CD8 T cell proliferation

49. Comparison of Antigen Presentation Abilities of DCs and Macrophages

50. Functional Features of Dendritic Cells Potency Small numbers of DCs pulsed with low doses of antigen stimulate strong T-cell responses. Primary responses Naive and quiescent T cells can be activated with antigens on DCs. Physiology CD4+ T helpers and CD8+ T killers are primed in vivo.

51. Differentially expressed genes during DC maturation mostly encode enzymes and protein involved in the control of: 1) cell cycle 2) cytoskeleton rearrangements 3) antigen processing 4) inflammation 5) apoptosis

52. NK cells activation Immature DC MIP-1  MIP-1  MIP-2 Mature DC Early-activated DC IL- 12 CD4 + T cell priming Apoptotic DC IL-1  TNF-  RANTES MIP-1  MCP-1 Immature DC Transitional DC Innate immune response 0 h2 h4 h6 h8 h18 h Time actin cytoskeleton rearrangments actin cytoskeleton rearrangments MIAP1 MIAP2 TRAF1 TRAF2 Adaptive immune response CD4 + T cell priming CTL priming CD40 B71/2 Peptide+MHCI/II increase of DC processing efficiency IL6 IL10 INFECTIOUS NON-SELF

53. DC T cell CD40L CD40 receptor-mediated endocytosis antigen processing pathways MHC II TCR CD28 B7-1/2 phagocytosis DC-T interactions after antigen uptake fluid-phase-endocytosis macropinocytosis NF  B activation MHC I soluble mediators cytokine receptors CD95 (Fas) FasL Live bacteria Toxins OX40L OX40 IMMUNE SYNAPSIS IL2

54. Kinetic of a bacterial-induced DC transcriptome: functional clustering of differentially expressed genes INFLAMMATION / CHEMOKINES / CYTOKINES Increasing expression 0 h 4 h 6 h 12 h 18 h 24 h 48 h AMYLOID A TGF  Flt3 IL6 MIP-1  RANTES IL12p40 MIP-1  MIP2 IL1  IL1RA TNF  EGF-BP C1qC C1qB C1q  MCP5 PDGF LT  JE mvrf186 0 h 4 h 6 h 12 h 18 h 24 h 48 h TGF  1 C3 IL15 IL10 0 h 4 h 6 h 12 h 18 h 24 h 48 h LT VEGF IP-10 MCSF IL12p35 GM-CSF 0 h 4 h 6 h 12 h 18 h 24 h 48 h IL2 Nature - Imm. 2, 882-888, 2001

55. 0 100 200 300 01020304050 mBM-DC Macrophages hours IL-2 is produced by BM-derived DC following bacterial activation but NOT by macrophages pg/ml Nature - Imm. 2, 882-888, 2001

56. 900 700 500 300 100 010 20 30 405060 BMDCsRAG2-/- TNF  IL-1  IFN  TNF  +IL-1  +INF  010 20 30 4050 60 010 20 30 405060 160 120 80 40 180 140 100 60 20 hours D8D1 D8BMDCsRAG2-/- 020 4060 1000 800 600 400 200 D1 CpG LPS LTA CpG ctrl 020 4060 400 300 200 100 PGN 120 80 40 0 1020 30 pg/ml ZYMOSAN D1 D8 BMDCsRAG2-/- 020 4060 5000 YEAST 25000 15000 020 4060 8000 6000 4000 2000 0 010 20 30 405060 160 120 80 40 UNTREATED IL-2 production by DC J. Immunol 2003

57. DCs produce IL-2 after contact with microbial stimuli but not after contact with inflammatory cytokines DCs are able to discriminate between a cytokine-mediated inflammatory process and the presence of infections

58. Microbial-MP’s Receptors Chemokines Cytokines NK T cell activation CD4 CD8 Immature DC INNATE response ADAPTIVE response Chemokines Cytokines NKT IL2 Trends in Immunol.23(4) 2002 IL-2 is a T, NK and B cell growth factor DC-derived IL-2 may be relevant for NK, T and B cell activation IFN 

59. NK cells activation Immature DC MIP-1  MIP-1  MIP-2 Mature DC Early-activated DC IL- 12 CD4 + T cell priming CTL priming Apoptotic DC IL-1   TNF-  RANTES MIP-1  MCP-1 Immature DC Transitional DC Innate immune response maturation time IL2 0 h2 h4 h6 h8 h18 h Time actin cytoskeleton rearrangments actin cytoskeleton rearrangments MIAP1 MIAP2 TRAF1 TRAF2 Adaptive immune response CD4 + T cell priming CTL priming CD40 B71/2 Peptide+MHCI/II increase of DC processing efficiency IL6 IL10 IL2 Gram- Gram+ Parasites INFECTIOUS NON-SELF IL2 yeast

60. DC are involved in the tolerization of peripheral T cells