Diseases of Immunity CL Davis General Pathology. Paul W. Snyder, DVM, PhD Experimental Pathology Laboratories, Inc

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1 Diseases of Immunity 2017 CL Davis General Pathology Paul W. Snyder, DVM, PhD Experimental Pathology Laboratories, Inc

2 Acknowledgements Pathologic Basis of Veterinary Disease, 6 th Ed Veterinary Immunology, An Introduction 8 th Ed Immunology, 6 th Ed The Immune System, Parham, 3 rd Ed Immunobiology, Janeway, 7 th Ed Snyder

3 Immunology The study of the immune system and of diseases that occur as a result of inappropriate or inadequate actions of the immune system.

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7 The body s defense system pathogen Physical and chemical barriers - epidermis, mucosal epithelium - ph of the stomach - mucociliary apparatus - lysozyme Innate immunity Adaptive immunity Snyder

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9 Innate Immunity Cells Phagocytic cells Macrophages and neutrophils Innate lymphoid cells Natural Killer (NK) cells Misc. ILC s Dendritic cells sense danger Not a unique population but rather a large collection of subpopulations Mature into different functional profiles dependent on the nature of the stimulus Snyder

10 Phagocytic Cells Macrophages Inflammatory mediators Mediated by TLRs TNF-α, IL-1 and IL-6 Chemokine IL-8 Phagocytosis Mediated by phagocytic PRRs Phagocytosis of senescent or apoptotic cells does not induce mediators immunologically silent Antigen presentation Co-stimulatory signal Neutrophils Recruited and activated by macrophage and mast cell products 1/17/2017 Snyder

11 Macrophages Snyder

12 Neutrophils Snyder

13 Neutrophil migration IL-8 (CXCL8) C5a LTB4 Snyder

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15 Function Cytotoxicity Direct Indirect ADCC NK Cells Perforin & granzyme, granulysin, fragmentin FasL (CD95) Cancer cells and virally infected cells 2 subsets in humans NK1 IFN γ macrophage activation NK2 IL13 inhibits macrophage activation Snyder

16 Receptors Activating C type lectin-like (KLR) NK Cells Recognize viral and stress induced proteins Inhibitory Killer cell Immunoglobulin-like receptors (KIR) FcγIII receptor (CD16) ADCC FasL TLR 3 & 9 Species differences in MHC I specific receptors on NK cells KIR cattle, pigs, dogs, cats KLR mice, rats, horses Inhibitory vs activating differences Snyder

17 NK Cells Regulation Activated by Type I Interferons ( & β) during viral infections IL-12 from activated macrophages & DCs activate NK cells and enhances killing Activated NK cells produce IFN γ facilitating a T H 1 IL-2 & IL-15 activate NK cell proliferation IL-21 down regulates NK cells NK T cells Properties of CTL & NK cells Early T H 2 late T H 1 NK dendritic cells Snyder

18 NK cells 5-15% of PBMC CD16 (Fc III R) CD56 CD2 IL-12 stimulates NK cells to produce IFN T H 1 Snyder

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20 Dendritic Cells Distinct morphology Not a unique population but rather a large collection of subpopulations located in lymphoid and nonlymphoid tissues Motility Specialized functions that shift upon activation Antigen capturing T Lymphocyte sensitizing Snyder

21 Dendritic Cells 2 Main DC subsets Conventional ( myeloid ) DC Plasmacytoid DC Anti-viral immunity TLR 7 & 9 Single stranded RNA (TLR7) and DNA viruses (TLR9) Type I IFN ( and ) Snyder

22 Dendritic cells Langerhans cell Interstitial DC Interdigitating DC Plasmacytoid DC Circulating DC Snyder

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24 Dendritic cells Important antigen presenting cells high expression of MHC II molecules efficient in processing and presentation of antigen Examples: Langerhans cells in the epidermis Interstitial (eg. dermal) dendritic cells Interdigitating dendritic cells in the paracortex of the lymph node and in the thymus Follicular dendritic cells in lymphoid follicles not bone marrow derived do not process antigen Paul Langerhan Snyder

25 Antigen sampling by DC s Macropinocytosis sampling the extracellular fluid Receptor-mediated endocytosis, clathrin coated pits Fc receptors C receptors (CR3 and CR4) C-type lectin receptors (CLR s) Scavenger receptors Phagocytosis Snyder

26 Antigen sampling by DC s DC cell ability to capture antigen is attributed to: High endocytic capacity Anatomic location Skin Mucosal surfaces Spleen Unique strategy for sampling through epithelial barriers CX 3 CR1, receptor for fractalline a chemokine on the surface of intestinal epithelium Snyder

27 DC activation Sense danger signals PAMPs PRRs TLR s and CLR s Mature into different functional profiles dependent on the nature of the stimulus Intrinsic bias of DC subpopulations even though very plastic Main LN homing chemokine receptor CCR7 which recognizes CCL19 (interdigitating DC) and CCL21 (stromal cells) expressed in T cell areas Snyder

28 DC activation Maturation process expression of MHC-peptide complexes expression of CD40, CD80 & CD86 Cytokine synthesis Activate naïve T lymphocytes - APCs Signal 1 = TCR - MHC/antigen peptide Signal 2 = CD28 - B7 Cytokines IL-12 T H 1 response, NK cell activation IL-4, IL-5, IL-13 T H 2 response Snyder

29 DC & NK Cell Cross-talk NK cell role in process of DC maturation NK cells kill DC that do not acquire a mature phenotype Death receptor-mediated not granule exocytosis NK cells stimulate maturation of DC s TNF IFN Specific action seems to be influenced by NK:DC ratios High ratio = kill Low ratio = stimulate maturation Snyder

30 Follicular dendritic cells Stromal cells (not bone marrow-derived) Do not express MHC II Do not process antigen Display antigen in the form of immune complexes Attract B cells to lymphoid follicles via CXCL13 Produce cytokines that influence B cell maintenance Snyder

31 Dendritic cell heterogeneity (mouse) CD4 -, CD8 + CD4 +, CD8 - CD4 -, CD8 - LC-type CD4 -, CD8 lo Spleen 23% 56% 20% Mes. LN 19% 4% 63% Skindraining LN 17% 4% 37% 33% Snyder

32 Innate Lymphoid Cells (ILCs) ILCs (non-t or B cells) develop in an Id2-dependent pathway, additional transcription factors drive additional subsets NK cells cytotoxic and non-cytotoxic subsets Helper cells (IL-25 IL-33 induces) ILC2 cells IL-5 & IL-13 anti-parasite responses ROR t cells (require IL-7) Functions: Lymphoid tissue-inducer cells (LTi) LN formation ILC17 cells IL-17A neutrophils extracellular bacteria ILC22 cells IL-22 extracellular bacteria Responses to infectious organisms Lymphoid tissue formation Tissue remodeling following Snyder 2017damage 32

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35 Innate Immunity Polarized Responses Similar to CD4+ T H subsets ILC2 promote T H 2 responses and inflammation at mucosal surfaces in conjunction with epithelial cells ILCs can be polarized toward restricted cytokine profiles - plasticity Exogenous signals drive the plasticity Disease associations: Allergic diseases Inflammatory diseases like IBD Autoimmune diseases Snyder

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37 Recognizing first responders RNA sensing Noninflammasome NLRs Regulation of adaptive immunity by innate immunity NLRP3 inflammasome a sensor for metabolic danger Snyder

38 Innate Immunity -Components Recognition molecules Pattern Recognition Receptors (PRRs) An ability to sense danger by cells other than T and B lymphocytes Pathogen Associated Molecular Patterns (phagocytic) Damage Associated Molecular Patterns (sensor) PRRs Toll-like receptor (TLR) family Nucleotide oligomerization domain-like receptor (NLR) family Retinoic acid-inducible gene I-like receptor (RLR) family Snyder

39 Innate Immunity - Components PRRs initiate signaling Initiate cascades for cytokine synthesis through transcriptional regulation and posttranslational modifications Type 1 IFNs (IFN-α and IFN-β) IL-1 family cytokines (IL-1β and IL-18) Inflammasomes intracellular sensors Cytosolic multi-protein complexes activate caspase-1 cleave pro-inflammatory cytokines into active forms Snyder

40 Pattern Recognition Receptors PRR s are classified as secreted, transmembrane and cytosolic forms Collectins and pentraxins are examples of secreted PRR Bind microbial surfaces and activate complement TLR s and C-type lectins are examples of transmembrane PRR s Snyder

41 Pattern Recognition Receptors Cytosolic PRR s Widely distributed, all nucleated cells Examples are: Retinoic-acid-inducible gene-1-like receptors (RLRs) Nucleotide oligomerization domain-like receptor receptors (NLR s) PRR s sense danger or stranger Initiate adaptive immune responses and regulate cell death (apoptosis) Snyder

42 Pattern Recognition Receptors Cell-intrinsic recognition mechanisms Involve Type I Interferons Three members: Retinoic acid inducible gene-1 (RIG-1) Melanoma differentiation associated gene 5 (MDA-5) Laboratory of genetics and physiology gene 2 (LPG-2) Specifically detect RNA molecular patterns not normally present in the cytoplasm Cell-extrinsic recognition mechanisms Allows uninfected cells to participate in immune responses Snyder

43 Pattern Recognition Receptors Members of the NLR are central regulators of immunity and inflammation Activate transcription factors like Nuclear factor κb (NF- κb) Interferon regulatory factor (IRF) Nuclear factor of activated T cells (NFAT) Some NLR members form multi-protein complexes referred to as inflammasomes that activate caspase-1 Caspase-1 cleaves pro-inflammatory cytokines like IL- 10 and IL-8 Other NLR members are involved in inflammasomeindependent innate immune responses Snyder

44 NLR proteins signal through different multicomponent signalsomes Snyder

45 Toll-like receptors (TLR) Expressed on numerous cell types sentinel cells Macrophages, mast cells, DC s, mucosal epithelium, hematopoietic stem cells Constitutive and inducible forms Link innate and adaptive immunity TLR can partially substitute for T cell activation of B cells Patients with immunodeficiency disease can produce limited quantities of antibody MyD88 KO mice have impaired IgM, IgG1 & IgG2c, but not other isotypes Snyder

46 Snyder

47 Toll-like receptors (TLR) TLR4 KO mice have impaired IgM responses Species differences in ligand specificity Genetic polymorphisms within a species 15 genes identified Innate autoimmunity HSP, fibrinogen, DNA RA and SLE Snyder

48 MyD88 TRAF6 MAPK Toll-like Receptors TIR = toll/il-1 receptor MyD88 an IL-1 receptor associated kinase (IRAK) a universal activator of NK-kB Tumor necrosis receptor-associated factor 6 (TRAF6) Signal 1 - transcription Signal 2 inflammasome activation of caspase-1 Pro-inflammatory mediators COX-2 PGE 2 NOS2 NO Caspase-1 pro-tnf TNF pro-il-1 IL-1 pro-il-6 IL-6 Snyder

49 Innate Immunity - Functions Phagocytosis Phagocytic cells and opsonins (C3b, PAMPS) Inflammation Mediators (C3a, C5a, inflammatory cytokines) Regulation Cytokines polarize acquired immune responses Interactions Cross-regulation within innate immunity and to acquired immunity Snyder

50 PAMPs DAMPs LUNG Abestosis COPD Asthma SKIN Allergy BRAIN Alzheimer s Multiple Sclerosis JOINTS Rheumatoid Arthritis Gout Inflammasomes Pro-IL-1 IL-1 Inflammatory Conditions HEART Hypertension METABOLISM Type II Diabetes CANCER Mesothelioma INTESTINE Inflammatory Bowel Disease Snyder

51 Innate (non-specific) immune system Controls infections during the time (5-7 days) that is needed to engage the adaptive immune system Activates and directs the adaptive immune system, primarily through signals delivered by dendritic cells. Snyder

52 pathogens viruses bacteria parasites physical and chemical barrier infection tissue damage innate immune system dendritic cells neoplasms adaptive immune system Snyder

53 Adaptive Immunity Acquired immunity or specific immunity Activation of a functional immune system Slow response Highly antigen Specific Improves during the response Memory Self renewal and clonal expansion Innate immunity or non-specific Rapid response Invariant Limited specificities No self renewal or clonal expansion

54 Adaptive Immunity Developed as a result of acquisition of the recombinase activating gene (RAG) Somatic recombination of gene segments that encode antigen receptors Distinction between innate and adaptive immunity is related to mechanisms and targets of recognition Innate relies on germ-line encoded receptors to a limited number of pathogen structures Adaptive can recognize an almost unlimited number of antigens because of somatic recombination generating antigen-specific receptors

55 Acquired Immune System Lymphoid cells T and B lymphocytes Antigen recognition molecules TCR, sig (BCR), MHC molecules Effector functions B cells Ig producing plasma cells CD8+ Cytotoxic T cells CD4+ T cells Helper T cells (T H ) Regulatory T cells (T reg ) T H 17 Interactions Polarized responses based on cytokine profiles

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57 Recognition molecules of the adaptive immune system Snyder

58 B and T lymphocytes B lymphocyte T lymphocyte specificity immunoglobulin T cell receptor maturation function fetal liver bone marrow ileal Peyer s patch bursa of Fabricius immunoglobulin secretion (plasma cells) thymus regulation cytotoxicity Snyder

59 Hematopoiesis hemopoietic stem cell interleukin-3, GM-CSF IL-7 erythrocytes B T lymphocytes platelets macrophages granulocytes Snyder

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62 Hematopoiesis Leukocytes programmed cell death Snyder

63 live cell neglect - lack of receptor stimulation or growth factors activation of TNF-receptor or Fas by ligand bcl-2 mitochondrial damage caspases breakdown of proteins apoptosis Snyder

64 T lymphocytes TCR TCR d CD4 + CD8 + T helper (T H ) cytotoxic T cell (CTL) Snyder

65 T lymphocytes TCR TCR d CD4 + MHC II CD8 + MHC I T helper (T H ) cytotoxic T cell (CTL) Snyder

66 Cytotoxic function of CD8 + T cells Snyder

67 Helper function of CD4 + T cells Snyder

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74 Exotoxins V regions of TCR & MHC II 5-20% of T cells Cytokines TNF, IL-1 Toxic-shock syndrome Superantigens Snyder

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76 Lymphoid Tissues Primary lymphoid tissues Generation of B and T lymphocytes Antigen-independent proliferation Include Thymus Fetal liver, bone marrow Secondary (peripheral) lymphoid tissues Initiation of antigen-specific immune response Antigen-dependent proliferation Include: Spleen (white pulp) Lymph nodes Peyer s patches & lymphoid nodules NALT, tonsils, BALT Snyder

77 Development of Immune System in the calf Snyder

78 Thymus Death by neglect >80% Negative Selection 5-10% Positive Selection <5% HSC ( cells/day) DN DP medulla cortex CD4 CD8 Snyder (< 5%, 10 6 /day)

79 Lymph node Snyder

80 Lymphoid follicles Dark zone - B cell proliferation, somatic hypermutation Light zone memory cells, plasma cell precursors, isotype switching Mantle mature naïve B cells Snyder

81 Germinal center Proliferation of B cells Somatic mutation and selection of high affinity B cells Isotype switching Memory cell induction Snyder

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85 Bursa of Fabricius Snyder

86 Spleen Snyder

87 Structure of spleen Snyder

88 Dog Rat Storage spleen Thick capsule and many trabeculae Prominent smooth muscle Relatively poorly developed white pulp Dogs, cats, horses Defense spleen Well developed lymphoid tissue Less smooth muscle Rats, mice, humans Intermediate spleen Cattle, swine Snyder

89 Peyer s patches Johannes Peyer Snyder

90 Snyder

91 Peyer s patches Group I Ruminants, dogs & pigs Ileal PP Primary lymphoid organ Developed at birth and involutes at sexual maturity Only B cells Jejunal PP which persist Group II Rabbits and rodents Randomly distributed between IL and JE Develop after birth and persist Snyder

92 Intraepithelial lymphocytes IEL CD3 CD3 + (90%) TCR (30-50%) TCR d (30-75%) CD4 + CD8- CD4 + CD8 + (5-8%) (5-8%) CD4 - CD8 + CD4 - CD8 + CD4 - CD8 - (20-40%) (30-60%) (5-10%) Snyder

7/6/2009. The study of the immune system and of diseases that occur as a result of inappropriate or inadequate actions of the immune system.

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