Kent T. HayGlass, Ph.D. Canada Research Chair in Immune Regulation Professor and Head,

1. March 2007 Welcome to Immunology Kent T. HayGlass, Ph.D. Canada Research Chair in Immune Regulation Professor and Head, Department of Immunology 789 3509 Questions??? hayglass@ms.umanitoba.ca www.umanitoba.ca/faculties/medicine/units/immunology/teaching.html

2. Why would a pharmacist study immunology? • A. You might flunk this course if you don’t • B. It is very exciting • C. It will be your partner for your entire professional life

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

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 systems of direct relevance to pharmacists • Essential notes, your text chapter, and all PPT slides, on U of M website (Medicine.. Immunology .. Teaching )

6. Today • 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 ?

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) VIRUS- Polio PARASITE - Tapeworm

10. Essential definitions Antigen: (Ag) something that stimulates an immune response ~ 10 amino acids long. 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 receptor that specifically binds one Ag and not another. Example : IgG

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

12. Essential characteristics of an immune response • Self non-self discrimination • Specificity • A way of amplifying selectively particular immune responses • Diversification: converting one response into multiple types • 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

13. What do you want in an immune response?

14. The optimal immune response 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 • Characteristics: • Quick to develop • Ag non-specific • Multiple effector mechanisms—both cell mediated and humoral • 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)

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

19. Innate Adaptive

21. Complement: A defensive, innate immunity, enzyme cascade

22. “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…

23. Key characteristics of the specific immune response (adaptive immunity) • Highly targeted, specialized host defenses adapted for many, specific types of threats: Ag specific • The capacity to remember previous encounters with a given Ag results in ability to mount faster, stronger and more effective responses during subsequent encounters: Immunological memory • The capacity to co-evolve with pathogens, by generating a virtually infinite number of new Ag receptors to counter newly evolved viruses and bacteria: Adaptability

24. Overview of the adaptive immune response

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

26. Adaptive/ Acquired/ Specific Immunity Key characteristics: Specificity Memory Adaptability How the Ag-specific response accomplishes this is covered next time...

27. What are the major cell types you need to know?Cells of the immune response Objectives: • What are, and how do we identify, the key cells of the immune response? • Anatomy of primary and secondary lymphoid organs—how do they get around?

28. Your choice? • Budget cutbacks mean that you can only afford one immune cell ie. Ab producing, cytotoxic, phagocytic, helper … • You have to pick which one you want — what’s your call?

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

30. The stem cell: The “centre of it all” The stem cell can differentiate into many different cell types. How does it decide what to become?

31. 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 vs…) 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

32. Myeloid cells in innate and adaptive immunity Read your text so you know 4 -5 facts about each major cell type.

33. 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.

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

35. Dendritic Cell • Low frequency cells with a key role in controlling initiation of adaptive immunity • Phagocytic. • Mature DC with Ag: Turn on and direct T cells.

36. 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

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

38. Lymphocytes in specific immunity • Key players for specific immunity are lymphocytes: B and T cells • Each 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. • 1. recognize each Ag with great specificity • exhibit memory • Population displays adaptability

39. 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) 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

40. Key CD Ags to remember CD3 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 CD 19 and 20 are on B cells but not T cells. CD56 is on NK cells but not other types of lymphocytes.

41. B cell development: How do Ab producing cells mature? Stem cell  B cell in Bone marrow (primary organ) Two key goals for the system: Generate multiple Ag specific receptors (1 per cell) Enzymes that join Ab gene components together to get a functional Ab gene are error prone: introduction of random variability Delete self reactive B cells generated by accident

42. T cell development: Helper and cytotoxic cells Stem cell  T cell in Thymus (a different primary organ) Goals: Generate diverse repertoire of TcR s (1 receptor / cell again) Enzymes that join TcR gene components together to get a functional receptor gene are error prone: continuous introduction of random variability Delete self reactive TcR generated by accident

43. Epithelial cells of the thymus form a network surrounding developing thymocytes

44. T cell development: Overview of the big picture • 1. Developing T cells generate wide diversity of novel receptors • 2. Each interacts with surrounding cells that express “self” Ag • 3. Receptors on maturing T cells may: • not bind at all (death by neglect) • bind very strongly (death by negative selection to delete strongly autoreactive cells:protection against autoimmunity) • bind with intermediate affinity (positive selection) and mature for export • 99% of all maturing stem cells in the thymus die.

45. Ag RECOGNITION: Receptors that bind Ag • B cells, T cells and the MHC all developed independent ways to specifically recognize Ag: • different genes for the receptors, • different ways of generating diversity • different purposes in immune responses. • However, all Ag receptors have in common that they depend on the physical binding of small molecules (~10 amino acids in size) in Ag binding pockets similar to a baseball in a glove. • Comparison to catching a cantaloupe, pingpong ball or cat

46. Constantends • usually anchored into the cell membrane • provide structural support • determine the functions for that family of molecules • Variableends • the part of the molecule that forms the Ag binding • pocket that recognizes one Ag compared to another • Ag recognition based on physical fit into the “pocket”

47. Overview of Lymphocyte functions

48. B cell receptors • B cell receptor: Ab (also known as Ig or immunoglobulin). • Unlike for T cells, the B cell receptor Ab: • is secreted, often at high concentration. • is of 5 main families (isotypes / classes) • IgM, IgG, IgA, IgD, IgE

49. Ab isotypes • IgM • first produced in primary responses • 2nd most common serum Ab • Opsonization, activates complement, neutralizing Ab • IgG • Dominates memory (20) responses in serum • Opsonization, activates complement, neutralizing Ab • Transplacental transfer

50. Ab isotypes (continued) • IgA • major Ab at mucosal surfaces • in colostrum, tears, GI and respiratory secretions • Opsonization, activates complement, neutralizing Ab • IgD • Who knows? • IgE • parasite defense; immediate hypersensitivity • ~10,000x lower levels than IgG, even in allergics