1. Primary lymphoid organs : - Bone marrow - Thymus Secondary lymphoid organs: - Spleen - Lymphatic vessels - Lymph nodes - Adenoids and tonsils - MALT (Mucosal Associated Lymphoid Tissue) GALT (Gut Associated Lymphoid Tissue) BALT (Bronchus Associated Lymphoid Tissue) SALT (Skin Associated Lymphoid Tissue) NALT (Nasal Associated Lymphoid Tissue) LYMPHOID ORGANS !!

2. THE TWO ARMS OF THE IMMUNE SYSTEM Monocytes, Macrophages, Dendritic cells, Granulocytes, NK cells and Complement components B and T cells Monocytes, Macrophages, Dendritic cells, Granulocytes, NK cells and Complement components !!

3. Professional phagocytic cells macrophages neutrophyl granulocytes dendrtitic cells the phagocytosed cells or molecules may modify the functions of the cell phagocytosis followed by enzymatic degradation Professional antigen presenting cells macrophages B lymphocytes dendrtitic cells they express MHCII molecules the protein degradation products (peptides) can be presented to T lymphocytes by MHC molecules ! ! ! !

4. Cells of innate immune system: Macrophages:  Macrophages are constitutively present in tissues and recognize microbes that enter these tissues and respond rapidly to these microbes. Initiate the immune response These cells are phagocytes (eliminate the pathogens) Activate the innate immune response (by secreted proteins, called cytokines) Activate the adaptive immune system. Macrophages serve as APCs that display antigens to and activate T lymphocytes Dendritic cells are constitutively present in tissues and recognize rapidly microbes that enter these tissues. Initiate the immune response. They have phagocytic capabilities migrate to lymph nodes, and display microbial antigens to T lymphocytes,professional antigen presentimg cells (APC) Neutrophil granulocytes are phagocytes, the main function to eliminate the pathogens Appear only in the circulation under normal condition Main actors In inflammatory processes !!

5. Infection Phagocyte activation How do immunocytes communicate: Soluble mediators CYTOKINES & CHEMOKINES Soluble proteinsproduced by cells. They have strong effect on the function of other cells. Bit similar to hormones.

6. How do immunocytes communicate: Cell-cell interaction Cell-cell communication takes place commonly in all the phases of the immune response T CTL T B Y Antibody production Activation of accessory cells Dendritic cell macrophage Antigen presentation Target cell Cell killing

7.  The most important mediators of indirect cell communication in the immune system („hormones” of the immune system).  Act in low concentrations. Cytokines can affect in an autocrine way, in a paracrine way, or in an endocrine way  pleiotropic effect.  Cytokines can act by synergistic or antagonistic ways to each other. A given cell may by affected by many cytokines resulting in the same effect  redundant effect. -The responsiveness of the given cell is based on the expression of cytokine-specific receptors. THE MOST IMPORTANT FEATURES OF CYTOKINES !

8.  Cytokines can be devided into sub-groups by origin and functional properties. Functional groups: Inflammatory cytokines Direct the development and maturation of immune cells Direct activation and differentiation of immune cells

9. hormons cytokines chemokine interleukins interferons Categories of cytokines

11. General schema of receptor funtion

12. MOLECULES OF THE IMMUNE SYSTEM Most important receptors of the imune system receptors (BCR, TCR, MHCI, MHCII, PRR, etc.) Soluble molecules: cytokines antibodies complement components

13. Receptors responsible for the recogniton of pathogens in the immune system Caracteristics of innate immune system, macrophage, dendritic cells PRR Pattern recognition receptors Danger signal and Pathogen recognition mainly in the innate immun system B cellsBCR (B cell receptor)Antigen recognition of B cell T cellsTCR (T cell receptor)Antigen recognition of T cell All nucleated cells in human MHC (MHCI) Major Histocompatibility Complex Do not recognise pathogens, but present intracellular peptides required for T cell receptor professional antigen presenting cells: macrophages, DC, B cells MHCIIDo not recognise pathogens, but present extracellular peptides required for T cell receptor

14. INNATE IMMUNITY I

15. Physical and chemical barriers  pH of 3-4  Pepsin Stomach Skin  Tight junctions  Keratin layer  Antibacterial peptides; Defensins  pH of 5.5  Fatty acids Burns and susceptibility to infections!  pH of 3.8-4.5  Lactobacillus  Lactic acid Vagina Eye  Tear film (Oils, lactoferin, mucin and lyzosyme) Respiratory tract Impaired cilia movement (CF)!  Cilliary movement  Coughing, sneezing

16. Monocite / macrofage DC Mast cell Granulo- cites NK cell B cell T cell Comple- ment Recogni -tion Cell-cel (APC) Communi cation Soluble effector function

17. INNATE IMMUNITY Pathogen recognition  PRRs (TLRs, C type lectins, Mannose and Glucan binding lectins, NLRs and RIG-I helicases) Phagocytosis, effector functions Communication/ Antigen presentation   Intracellular – on surface MHC I complex proteins Extracellular – on surface MHC II complex proteins

18. Recognition is inevitable Innate immunity as a first line of defence Innate immune cells recognize frequently found structures of pathogens, these are not found in human cells! Examples: duple strain RNA bacterial cell wall components bacterial flagellin…. !!

19. Danger signal! The innate immune system also recognizes molecules that are released from damaged or necrotic cells. Such molecules are called damage-associated molecular patterns (DAMPs). !!

20. Recognition is inevitable Innate immunity as a first line of defence Innate immune cells recognize frequently found structures of pathogens, these are not found in human cells! Examples: duple strain RNA bacterial cell wall components bacterial flagellin…. !!

23. PAMPs- Pathogen associated molecular patters Structures on pathogens recognized by the innate cells

24. PRR types TOLL RIG like receptors NOD Scavanger receptors C type lectin receptors Mannose recognizing receptors

25. Macrophage/Dendritic cell TLR5 Flagellin Virus TLR3 dsRNA TLRs RECOGNIZE VARIOUS MICROBIAL STRUCTURES TLR2 Peptidoglycane Gram+ TLR4 LPS TLR6 Gram- Interferon producing cell PC/DC IFN  Bacteria CpG DNA TLR9 TLR7TLR8ssRNS

26. TLR receptors: Intracellular and cell surface sensors. Viral RNA, non-methylated DNA characteristic of bacteria, bacterial flagella, bacterial surface components (lipoproteins, peptidoglicans) and fungi structures. Partial overlapping recognition between NOD and RIG like receptors.

27. TLRsLigands:Microorganis m recognized: Cells carrying receptor: Cellular location: TLR1:TLR2 Lypopotreins lypoteichoic acid proteoglycan zymosam Bacteria Parasites DCs, mono, Eos/Baso, mast cells Plasma mem. TLR2:TLR6-”- G+ Bacteria Fungi-”-Plasma mem. TLR3dsRNAVirusesNK cellsEndosomes TLR4:TLR4LPSG- Bacteria M ϕ, DCs Plasma mem. TLR5FlagellinMotile BacteriaIntestinal Epi.Plasma mem. TLR7ssRNAViruses pDCs B cells Eos/Baso Endosomes TLR8ssRNAVirusesNK cellsEndosomes TLR9 Unmethylated CpG-ODN (ssDNA) Bacteria Viruses pDCs B cells Endosomes

28. NOD like receptors NOD-like receptors: Intracellular receptors. Recognizing intracellular pathogen and danger signals. Partial overlapping recognition with TLRs.

29. RIG receptors: Intracellular sensors. Recognizing viral RNA, inducing an anti-viral response. Partial overlapping recognition with TLRs.

30. Additional PRRs:

31. Eukaryotes Glucosamine Mannose Galactose Siallic acid Mannóz Prokaryotes Macrophage / Dendritic cell Mannose receptors Bacterium Mannose

32. direct connetion between innate cells and pathogen )( Specificity of innate immunity Few receptors (20-30) are responsible for the recognition of all the pathogens !

33. OPSONIZATION ! ! Main opsonins: antibodies Complement fragments Acute-phase proteins Opsonization facilitate and accelerate the recognition of the pathogen by phaogocytes, opsonins form a bridge between pathogen and a phagocyte connecting them.

35. Soluble mediators

36. Pathogen recognition by innate immune system 1.Directly via PRR 2.Indirectly via opsonization

37. INNATE IMMUNITY Pathogen recognition  PRRs (TLRs, C type lectins, Mannose and Glucan binding lectins, NLRs and RIG-I helicases) Effector functions, elimination of pathogens Communication/ Antigen presentation   Intracellular – on surface MHC I complex proteins Extracellular – on surface MHC II complex proteins

38. INNATE IMMUNITY II Effector functions, elimination of pathogens 1.Phagocytosis 2.Killing with soluble mediators 3.Complement system 4.NK cell activation !!

39. Degradation ACTIVATION Uptake PHAGOCYTOSIS Phagocyte PRR 0.5 - 1 hours The amount of internalized particles is limited Bacterium Intracellular killing Antigen presentation T cell ACQUIRED IMMUNITY

40. THE PHAGOCYTIC SYSTEM MACROPHAGES DENDRITIC CELLS NEUTROPHILS

41. Phagocytic cells -Macrophages -Dendritic cells -Neutrophil granulocytes (No presentation on MHC II) Professional antigen presenting cells -Macrophages -Dendritic cells - B lymphocytes (no killing action, only Ag presentation)

43. PHAGOCYTOSIS

44. Extracellular pathogen phagocytosis and killing

45. Extracellular pathogen phagocytosis and killing

46. Soluble mediators reeased from macrophages, granulocytes are responsible for kiliing of extracellular pathogens ROS reactive oxigen species NO nitric oxide Destructive enzymes, antimicrobial substances 2.

47. Intracellular bacterial evasion of killing in phagocytes Defensins Phagosome acidification Phagosome–lysosome fusion Lysosomal enzymes Intraphagolysosomal killing ROI RNI Iron starvation Tryptophan starvation Macrophage effector capacity

48. Lysis of bacteria COMPLEMENT ACTIVATION Inflammation Chemotaxis Complement-dependent phagocytosis Bacterium COMPLEMENT Lectin pathway Alternative pathway Antigen + Antibody ACQUIRED IMMUNITY Complement-proteins Few minutes – 1 hour Enzymes get fragmented, complement activity can be exhausted 3.

49. NK cells Major differences between NK cells and B/T lymphocytes:  Contain large cytoplasmic granules.  Responds fast, circulate in a partly activated state.  Do not express surface receptors produced by rearranged genes.  Have a range of cell-surface receptors that deliver activating or inhibitory signals  Have two main types of receptors: Ig-like Rs and the Lectin-like Rs (inhibitory and activating) that recognize altered cell surface proteins as a result of a virus infection.  Overall balance of inhibitory or activating signals decides if the NK cell killing action will take place.  Individual NK cells express different combinations of receptors- heterogeneity  repertoire of responses to pathogens.

50. Killing of the cells infected with intracellular pathogens KAR KIR KIR – Killer Inhibitory Receptor association to MHC I KAR – Killer Activatory Receptor NK Target MHC+ NK KAR KIR Target MHC- ! 1.The activity of NK cells is enhanced by activatory receptors 2.Inhibitory receptors block NK cell activity. Self cells are protected by inhibitory receptors. 3.Infection or tumors may increase the amount of activation and/or decrease the efficacy of inhibition Inhibition of lysis lysis

51. Adaptive components are also able to activate NK cells ADCC-Antibody Dependent Cell Cytotoxicity Activating NK cells through FcR on NK cells recognizing pathogen-bound Antibodies

52. Lysis of infected cell ACTIVATION OF NATURAL KILLER CELLS Kinetics of the activity of the complement system and NK cells in virus infection IFN  IL-12 Complement system NK-cells days Relatív szint/aktivitás NK-CELLS Virus-infected cell PRR RECOGNITION ACTIVATION RECOGNITION OF ALTERED HOST CELLS

53. RECOGNITION RECEPTORS, SIGNAL TRANSDUCTION, PHAGOCYTOSIS, EFFECTOR MECHANISM Pattern recognition Receptors (PRRs) Engagement of PRRs triggers phagocytosis and cytokine production

54. INNATE IMMUNITY Pathogen recognition  PRRs (TLRs, C type lectins, Mannose and Glucan binding lectins, NLRs and RIG-I helicases) Phagocytosis, effector functions Communication/ Antigen presentation   Intracellular – on surface MHC I complex proteins Extracellular – on surface MHC II complex proteins

55. Recognition of PAMP or DAMP induce inflammation

56. NK-cell IL-12 macrophage IFN  cytokines neutrophil TNF-  INFLAMMATION – ACUTE PHASE RESPONSE hrs Plasma level LPS (endotoxin) (Gram(-) bacteria) TNF-  IL-1  IL-6 Kinetics of the release of pro- inflammatory cytokines in bacterial infection TNF-  IL-1  IL-6 Few hours ACUTE PHASE RESPONSE Bacterium LPS DANGER SIGNAL ACTIVATION PRR Reminder

58. INNATE IMMUNITY Pathogen recognition  PRRs (TLRs, C type lectins, Mannose and Glucan binding lectins, NLRs and RIG-I helicases) Phagocytosis, effector functions Communication/ Antigen presentation   Intracellular – on surface MHC I complex proteins Extracellular – on surface MHC II complex proteins

59. INTERFERON RESPONSE Besides virus infected cells.. Plasmacytoid dendritic cells (pDCs) produce 1000x more type I interferon than other cells NATURAL INTERFERON PRODUCING CELLS – IPC After viral infection they are accumulated at the T cell zone of the lymph nodes

60. EFFECTS OF TYPE I INTERFERONS vírus Activate cellular genes that destroy viral mRNA and inhibit translation of viral proteins NK cells have a basal cytotoxicity level that is increased up to x100 fold with the exposure to type I IFNs and produce IFN-γ when exposed to IL-12 Helping the initiation of effector T cells- adaptive immune response.

61. paracrine autocrine Infected cell subtypes IFN-  IFN-  IFN response IRF-3 IRF-7 Virus IFN-  IFN-  NF  B AP-1 Type I IFN receptor IFN response VIRUS INDUCED TYPE I INTERFERON PRODUCTION IRF: interferon regulatory factor IRF-3

62. TRIF TANK IKKεTBK1 IRF-3 TRIF TRAM TLR3 TLR4 MyD88 IRF-5 TLR7 TLR8 TLR9 IFN-β, IFN-α1 RIG-1 Stimulation of Ig-production by B-cells Type I interferon receptor IRF-7 Increased cytotoxicity and proliferation of NK-cells Activation of  - and γδ T-cells Increased antigen presentation in myeloid dendritic cells IRAK-1 TRAF-6 IRF-7 MULTIPLE EFFECTS OF TYPE I INTERFERONS on immune cells

63. INTERFERON EFFECTOR PATHWAYS induction of the „antiviral state” 1. Mx GTPase pathway –block viral transcription 2. 2',5'-oligoadenylate-synthetase (OAS) -directed Ribonuclease L pathway –degrade viral RNA 3. Protein kinase R (PKR) pathway (Ser/Thr kinase, dsRNA- dependent) –inhibit translation, preventing viral protein synthesis 4. ISG15 ubiquitin-like pathway –modify protein function CELLULAR GENES THAT CONTROL ALL STEPS OF VIRAL REPLICATION

64. Plasma membrane Cytoplasm Type I. IFN receptor Type II. IFN receptor Type III. IFN receptor (IFNλ) TYK2JAK1TYK2JAK1 JAK2 JAK1 JAK2 STAT1 STAT2 Nucleus STAT1STAT2 P P STAT1STAT2 P P STAT1 P P P P IRF9 ISRE GAS – promoter elements Antiviral immunityAntimycobacterial immunity ISG15, Mx, OAS and PKR IL-10R2IFNLR1IFNAR1/2IFNG1/2 Interferon-stimulated genes Interferon-stimulated Regulatory elements ISGF-3 GAS: Gamma Activating sequence Signal Transducers and Activators of Transcription

65. Oligomer accumulation in cytoplasmic membranes (e.g. ER) (Nucleus) (Cytoplasm) ISRE MxA MxA monomer MxA oligomer Trapped viral components (Nucleus) (Cytoplasm) ISRE OAS1 Inactive OAS1 monomer Induction by viral dsRNA Active OAS1 tetramer synthetized pppA(2’p5’A)n inactive RNaseL monomer active RNaseL dimer cleaved RNA (Nucleus) (Cytoplasm) ISRE PKR Inactive PKR monomer Active PKR dimer Induction by viral RNAs EIF2  P Inhibition of translation Mechanism of action of MxA, OAS1 and PKR

66. NK-cell IL-12 macrophage IFN  cytokines neutrophil TNF-  INFLAMMATION – ACUTE PHASE RESPONSE hrs Plasma level LPS (endotoxin) (Gram(-) bacteria) TNF-  IL-1  IL-6 Kinetics of the release of pro- inflammatory cytokines in bacterial infection TNF-  IL-1  IL-6 Few hours ACUTE PHASE RESPONSE Bacterium LPS DANGER SIGNAL ACTIVATION PRR Reminder

67. RECEPTORS ON MACROPHAGES TLR4 + CD14 MHCI MHCII TLR – pathogen pattern CR1 (CD35) CR3 (CD11b/CD18) LFA1 (CD11a/CD18) Fc  RIII (CD16) Fc  RII (CD32) Fc  RI (CD64) Ag + IgG complex Mannose receptor Scavanger receptor

68. FcRs

69. RECEPTORLIGANDFUNCTION FcRIgG, IgEOpsonized phagocytosis, ADCC, release of inflammatory mediators CR3iC3B, ICAM-1Opsonized phagocytosis Macrophage Mannose Receptor LectinEndocytosis, phagocytosis, antigen capture and transport SR-ALPS, polianions, lipoteikolic acid Endocytosis, phagocytosis, adhesion CD14LPSTransduces LPS activation, TNFa release CCR1MIP1a, MCP-3Recruitment, migration of monocytes CCR3EotaxinHaematopoiesis, HIV-1 coreceptor CCR5MIP1Haematopoiesis, HIV-1 coreceptor CXCR4SDF-1aHaematopoiesis, HIV-1 coreceptor Receptors and molecules of macrophages

70. INNATE IMMUNITY Pathogen recognition  PRRs (TLRs, C type lectins, Mannose and Glucan binding lectins, NLRs and RIG-I helicases) Cell activation  Increase in MHC, co-stimulation, killing efficiency (ROI, NO, lysosomal enzymes) and cytokine secretion Phagocytosis and Antigen processing  Intracellular – degradation in Proteosomes Extracellular – Endiocytosis then degradation in Phagolysosomes Antigen presentation   Intracellular – on surface MHC I complex proteins Extracellular – on surface MHC II complex proteins