Jude Uzonna, DVM, Ph.D. Department of Immunology Phone: 977 5659

1. Welcome to March 2008 Immunology Jude Uzonna, DVM, Ph.D. Department of Immunology Phone: 977 5659 Email: uzonna@cc.umanitoba.ca www.umanitoba.ca/faculties/medicine/units/immunology/teaching.html

2. Why would a pharmacy student study immunology? • It is required (they made me do it) • You might flunk this course if you don’t • It is very exciting • It will be your partner for your entire professional life

3. Food for thought………. Why do some individuals infected with same pathogen fail to respond (recover) following specific anti-microbial therapy?

4. Immunology and Pharmacy • Value of Pharmaceuticals in isolation? • AIDS: triple therapy • Pharmaceuticals can be considered a partner to— not replacement for-- the immune system

5. Immunology module objectives: • Overview of key characteristics of innate vs acquired immunity • Who are the players? Key cells • How is immunity activated then controlled? • Applied immunology of direct relevance to pharmacists • Vaccines • Hypersensitivity reactions

6. Today’s Objectives: • Identify the major principles of the human immune response • Introduce the two main forms of immune response. What they are and how do they relate to one another • Key phases of adaptive immune response

7. The perfect world

8. The real world

9. Bacteria, Fungi, Parasites, Viruses BACTERIA -Clostridium difficile (causes antibiotic-associated colitis & antibiotic-associated diarrhea) FUNGUS -Epidermophyton floccosum (causes athlete’s foot) PARASITE - Tapeworm VIRUS- Polio

10. Essential definitions Antigen: (Ag) something that stimulates an immune response Can be any molecule: components of bacterial cell walls to exotic chemicals like poison ivy sap to your own proteins Antibody (Ab): a family of defensive proteins your body makes when it is stimulated by Ag. Ab contains a site that specifically binds one Ag and not another. Example : IgG

11. Essential definitions contd: Lymphoid organ: Anatomical site where immune cells and immune responses are generated Central or primary Lymphoid organs: Sites of generation of lymphocytes (bone marrow, thymus) Peripheral or secondary Lymphoid organs: Sites of adaptive immune responses are initiated and where lymphocytes are maintained (eg. spleen, lymph nodes)

12. Time to design your own defense system for a bank, security company, army, your personal computer,... What are the key characteristics of an effective defense system?

13. Essential characteristics of the immune system • Self non-self discrimination • Specificity • A way of amplifying selectively particular immune responses • Diversification: converting one response into multiple types • Regulation: Turning responses off so that they don’t get out of control • Memory • Redundancy • The ability to respond to a changing environment by inventing new Ag receptors

14. The optimal immune response has two components: One component that is quick to develop and Ag non-specific to contain the pathogen initially: INNATE IMMUNITY A second component that is Ag specific to allow you to select the most appropriate of the ~30 different types of immune responses you have available, keep them highly targeted, exhibit memory ... ADAPTIVE IMMUNITY

15. Overview of innate and adaptive immunity

16. Develop a system that can respond virtually instantly to readily identifiable potential pathogens>>> The innate immune response • Key characteristics of innate immunity: • Quick to develop • Ag non-specific • Multiple effector mechanisms—both cell -mediated and humoral components • Goal: Contain the pathogen in the initial hours and days of infection, giving more sophisticated defenses time to expand and be deployed.

17. Innate Immunity • Main Assets: • Rapid (minutes to hours for full activation) , covers the 4-10d for a primary response to develop • Intense (essential role in inducing a strong inflammatory response) Main Liabilities: • No adaptability to new stimuli, hence no protection from novel pathogens (ie. new flu variants) • No memory -- no capacity to “learn from previous infections” (an innate immune response is the same speed, type and intensity on the first or 10th exposure to a pathogen) • Poor regulation: self/nonself discrimination not be efficient resulting in collateral tissue damage • Poor amplification: Magnitude of response always same

18. How does it work? • 1. Constitutive • 2. Activated by “danger signals” (e.g. lipopolysaccharide) molecules widely conserved on pathogens • 3. Generation of inflammatory response • What is inflammation? 4 characteristics. • Major components of innate immunity • Cell-mediated: • Phagocytic cells (macrophages, neutrophils) • NK cells (natural killer) • Humoral factors • Complement • Acute phase proteins

19. Major components of innate immunity Cell-mediated: • Phagocytic cells (macrophages, neutrophils) • NK cells (natural killer) Humoral factors: • Complement • Acute phase proteins • Interferons

20. Phagocytosis: Innate Adaptive

21. NK cell cytotoxicity:

22. Complement: A defensive, innate immunity enzyme cascade

23. “Innate” is all you need if you’re a frog and planning on a short lifespan with lots of babies... • The trade off for “quick to respond to widely expressed danger signals that bind to a small family of receptors (~10)” is an absence of : • Ag specificity • Specialization • Adaptability to new pathogens • Hence, we evolved an Ag-specific immune response…

24. Why adaptive immunity evolved? • Shortcomings of innate immunity: • Non-specific • Similar pattern of response for all pathogens • Poor regulation • Control mechanisms are poor or lacking • Poor amplification • Response magnitude same for all insults • Lack of self discrimination • Harm to self results for lack of specificity • Short duration • No memory

25. The enemies are different… BACTERIA -Clostridium difficile (causes antibiotic-associated colitis & diarrhea) FUNGUS -Epidermophyton floccosum (causes athlete’s foot) PARASITE - Tapeworm VIRUS- Polio

26. ….therefore responses must be tailored for specific enemies. • Successful immune response is a huge investment!! Hence: you need to remake it: •  Faster •  Larger •  More specific •  Less damaging to self

27. Key characteristics of the specific (adaptive) immune response

28. Mediators of adaptive Immunity • Highly dependent on T and B cells bearing specific receptors • T cell receptors • Membrane immunoglobulin on B cells • Other innate cells play some role, but T and B cells are the most important • Details in Lecture 2

29. Clonal selection • Each lymphocyte bears a single receptor with unique specificity • High affinity interaction of a lymphocyte with its specific antigen leads to lymphocyte activation • The activated lymphocyte proliferates and produce daughter cells (clones) each bearing receptors identical to the parental cell • Lymphocytes with receptors specific for ubiquitous self antigens are deleted at the early stage of development

30. The clonal selection of lymphocytes

31. Phases of adaptive immune response

32. Immunologic Memory: • Ability of the immune system to respond more rapidly and effectively to pathogens that have been encountered previously – either by previous infection or by vaccination • This reflects the pre-existence of clonally expanded lymphocytes with specificity for that antigen. • Hallmark of adaptive immunity

33. Schematic representation of memory response

34. Effectiveness of memory: • More responder cells available • Frequency higher than naïve cells • More efficient antigen recognition/activation • May not require costimulatory signals for activation • Rapid and more effective migration to tissues and lymph nodes • Express different homing/chemokine receptors • More effective function • Produce qualitatively and quantitatively more cytokines (T cells) or antibodies (B cells) than naïve cells • Longer lasting

35. Evading the immune response: Dirty tricks our enemies play • Concealed Ag (bacteria) • Shed Ag (parasites) • Changing Ag (flu, parasites) • Manufacture immunosuppressive • molecules (pox viruses)

36. Last Lecture Recall • Key characteristics of the immune system: • Two key components of immunity • Innate and Adaptive • Shortcomings of innate immunity • Characteristics of adaptive immunity • Both innate and adaptive components are important for optimal immunity • Pathogens can trick the immune system

37. Immunity is like government • Canada: Three tiers • Federal, Provincial, Municipal • Immunity: Two tiers • Innate, Adaptive • Government: • Key players (prime ministers, premier, mayors etc • But all levels must integrate for efficient governance!!

38. Cells, cellular integration and immunity Objectives: • What are, and how do we identify, the key cells of the immune response? • Antigen recognition by B and T cells • Anatomy of primary and secondary lymphoid organs—how do they get around? • Central and Peripheral Tolerance • Self/non-self discrimination

39. Many cells from one… (the centre of it all….)

40. The stem cell can differentiate into many different cell types. How does it decide what to become?

41. Stem cell career counseling Two major influences shape stem cell differentiation: 1. ENVIRONMENT in which it develops (which primary lymphoid organ--ie bone marrow vs thymus 2. the CYTOKINES AND GROWTH FACTORS they are exposed to during this process Primary lymphoid organs are the sites at which the cells of the immune response develop to get ready for later Ag/Danger signal –mediated activation

42. Neutrophils • Most common WBC in circulation, hallmark of inflammation (“polymorphs”) • Named for staining properties (granules) • First line of cellular defense: acts on bacterial, viral infections. Rapid release but short lived • Highly phagocytic, digestion • Produces toxic mediators.

43. Macrophage (monocyte) • First phagocytic cell discovered • BM derived, circulate 2d, then reside in tissue, organs, long term • Phagocytosis (via innate receptors and opsonization), • Degrade ingested particles and Ag presentation for T cell activation • Release regulatory and toxic molecules

44. Dendritic Cell • Low frequency cells with a key role in controlling initiation of adaptive immunity • Phagocytic (immature more than mature) • Process (degrade) antigens into peptides) • Present peptides (loaded on MHC molecules) to T cells • Deliver costimulatory signals to T cells • Mature DC with Ag: Turn on and direct T cell response

45. Basophil, Mast Cell, Eosinophil • Three separate families of granulocytes distinguished by their physical appearance • All are important in anti-parasite immunity and… • Can be phagocytic but most important mechanism of action… degranulation • All important in allergy

46. Myeloid cells in innate and adaptive immunity: overview

47. Lymphocytes are mostly small and inactive cells But watch out once they’re activated!

48. Lymphocytes in specific immunity Key players for specific immunity are lymphocytes: B and T cells Each lymphocyte binds only a single antigen, but your immune system has billions of specificities (using only one receptor per cell), so the systems as a whole can recognize billions of different Ags. Use ~30 different effector mechanisms All aimed at destruction or localization of pathogens.

49. How do you tell different cell types apart? Physical appearance:Lymphocytes small, granulocyte larger with granules that stain in different ways with dyes used in lab. (Differential cell count) Cluster of Differentiation (CD) Ag system:~150 cell surface proteins distinguished with Abs used as a diagnostic tool. Allows us to positively identify different cell types, function, state of activation. Memory work is SOOOO boring , but

50. Key CD Ags to remember CD3 present on all T cells, NOT on B cells. Among T cells there are two main sub-groups: CD4 “helper T cell” CD8 cytotoxic T cell CD19 and CD20 are on B cells but not T cells. CD56 is on NK cells but not other types of lymphocytes.