IMMUNOLOGY MODULE 1 FALL 2013

DAVID ALLMAN, Ph.D., Course Director

CONTACT INFORMATION: Phone: Email: 215-746-5547 (office) 610-209-5689 (cell) dallman@mail.med.upenn.edu

This document contains: 1. A description of the course and schedules for lectures and small group sessions. 2. Advanced lecture notes for all lectures dealing with fundamental immune mechanisms. 3. Sample exam questions.

IMMUNOLOGY LECTURES HELD IN REUNION HALL, JOHN MORGAN BUILDING COURSE GOALS: The goals of this course are several: First, we will introduce you to the basic principles and concepts in immunology. Second, we will work with you to develop your understanding of these concepts, such that you should acquire a strong sense of how the immune system works and how to recognize diseases associated with immune system function or dysfunction. Third, you will learn about assays and diagnostic assays that are developed through manipulation of the immune system. Finally, I hope you will leave the course with a foundation for the field of immunology which will enable you to learn more about the immune system on your own as needed throughout your clinical career. COURSE DESCRIPTION: The course will begin with a general overview of immunity, followed by in-depth considerations of the underlying cellular, molecular, and genetic events in immune system development and function. Afterwards, later lectures and discussions will focus upon more specialized clinically related issues in immunology, such as disease states involving the immune system. To these ends, the course will consist of formal lectures coupled with several informal small group discussion sessions. The latter sessions will be an opportunity for each of you to learn with and from your colleagues about clinical case studies that reveal fundamental concepts in immunology. In these sessions you will also work with your colleagues to answer 1-3 problems that serve as reviews of fundamental concepts discussed in previous lectures. EXAMS: There will one final exam. Both are in-class exams. I will divide the exam into two sections of multiple-choice questions. The first section will consist of multiple-choice questions designed to test your retention and understanding of basic facts and concepts in immunology. The second section will have fewer questions and these will consist of multiple choice or short answer questions that require problem solving. These latter questions will revolve around a description of clinical data and/or a controlled experiment. GRADING: Grading is pass-fail according to guidelines set by Suite 100. READINGS: Readings in the primary text are given in the Table on page 5. I strongly suggest you use these, as you see fit, to strengthen your grasp and broaden your perspective of the concepts conveyed in class. TEXT: The recommended text is Basic Immunology, Function and Disorders of the Immune System by Abul K. Abbas and Andrew H. Lichtman. You may also use additional resources such as the Janeways Immunobiology (8th edition) or The Immune System by Peter Parham (3rd edition).

LECTURE SCHEDULE
Mon 10/21 8-9 Introduction to Immunology: Concepts in adaptive and innate Immunity Antibodies as Antigen-specific Effector Molecules Anatomy of the Immune System The Cellular and Genetic Basis of Antibody Specificity B cell responses Innate Immunity I Innate Immunity II MHC & Antigen Presentation T Cell Development and Activation Tolerance and Autoimmunity Review Transplantation Immunology Vaccines Hypersensitivities Mechanisms of Inflammation Primary Immunodeficiencies Final Course Review Dr. Allman

9-10

Dr. Allman

Fri

10/25

10-11 9:30-11:30

Dr. Allman Dr. Allman

Tues 10/29

Tues 11/5

8-9 9-10

Dr. Allman Dr. Behrens Dr. Behrens Dr. Allman Dr. Allman Dr. Allman Dr. Allman Dr. Johnson Dr. Weiner Dr. Kambayashi Dr. Behrens Dr. Sullivan Dr. Allman

Fri Mon. Wed Fri Fri

11/8 11/11 11/15 11/19 11/22

10-11 8-10 8-10 11-12 8-9 10-11 9-10 9-10 10-11

Tues 12/3 Wed Fri 12/4 12/6

Thurs 12/12 Wed 12/18

8-9 8-10

Small group session and exam schedule:

Small Group Sessions
Wed 10/31 10:30-12:30 Thurs 11/8 8-10 Discussion 1 Discussion 2 Discussion 3 Joint (Microbiology and Immunology) Case Discussion 4 Discussion 5 Discussion 6 Discussion 7 Optional Review

Thurs 11/12 9:30-12 Wed 11/20 8-10 Thurs 11/21 8-10 Thurs 12/5 Tues 12/10 8-10 10-12

Tues 12/13 8-10 Tues 12/17 8:30-10

Exam (Dunlop Auditorium)

Thurs 12/19 12-2

Final exam

SUGGESTED READINGS PAGES IN ABBAS RD 3 EDITION Pages 1-13 Pages 70-74; Chapter 8 Chapter 1 Pages 79-84; Pages 179-189 Chapter 7 Chapter 2 Chapter 3 Chapter 4 Pages 84, 85; Chapter 5 Chapter 9 Chapter 10 Chapters 5 & 7 (review) Chapter 11 Chapter 2 (review) Chapter 12 PAGES IN ABBAS TH 4 EDITION Pages 1-13 Pages 74-78; Chapter 8 Chapter 1 Pages 82-88; Pages 172-184 Chapter 7 Chapter 2 Chapter 3 Chapter 4 Pages 88-90; Chapter 5 Chapter 9 Chapter 10 Chapters 5 & 7 (review) Chapter 11 Chapter 2 (review) Chapter 12

LECTURE 1 2 3 4 5 6, 7 9, 10 11 12 14 15 16 17 18

TOPIC Intro / Adaptive and innate immunity Antibodies as antigenspecific effectors Anatomy of the immune system Cellular & genetic basis for antibody specificity B cell responses Innate immunity I, II MHC and antigen presentation I & II T cell development and activation Tolerance and autoimmunity Transplantation immunology Vaccines Hypersensitivities Mechanisms of inflammation Primary immunodeficiencies

LECTURE 1 ADVANCE ORGANIZER: Most animals have protective systems to survive attack by foreign materials (pathogens, etc.). Some are "constitutive" mechanisms, serving to protect against any type of invasion, such as the keratinized layer of the epidermis and associated secretions. These are examples of innate mechanisms. Others, including the immune system of vertebrates, possess the capacity to mount responses that are INDUCIBLE - becoming active only after the offending agent is present. These protective mechanisms show different degrees of specificity. Innate mechanisms can be virtually nonspecific; i.e.; they act against any and all offending agents in the same fashion, while others show a moderate degree of specificity at best. By contrast, within the adaptive immune systems, many responses are highly SPECIFIC - possessing the ability to recognize subtle differences between different offending agents, tailoring the response to these differences. Finally, adaptive immune systems display two additional important properties: MEMORY, implying that upon a second or further exposure to a given foreign substance, the immune system responds more rapidly and more avidly; and NON-RESPONSIVENESS TO SELF, implying that structures which are a normal part of the host organism are not attacked and destroyed by its own immune system, even though they could be attacked by the immune system of another individual in which they were not "self". Significantly, adaptive immune responses follow predictable and distinct kinetics upon initial versus secondary exposure to a given antigen. These responses involve both an induction phase, before detectable immune activity is observed, and an effector phase in which immune cells and/or molecules activity work to eliminate the offending agent.

I. INTRODUCTION TO BASIC MOLECULAR AND CELLULAR IMMUNOLOGY A. PROTECTIVE MECHANISMS - GENERAL CONSIDERATIONS 1. PROTECTIVE MECHANISMS EXIST IN MOST ANIMALS. a. SPECIFIC vs NON SPECIFIC nonspecific mechanisms - not cognitive keratinized skin layer inflammation processes specific -requires recognition b. INDUCIBLE vs CONSTITUTIVE PROTECTIVE SYSTEMS constitutive - always active, require no stimulus inducible - require an activating stimulus (antigen) c. IMMUNITY AMONG VERTEBRATES B. IMMUNITY IS A PROTECTIVE MECHANISM 1. The notion of immunity is not new. That disease exposure could lead to subsequent disease resistance was recorded by the ancient Greeks. Accounts of deliberate exposure to disease-producing materials as a means of inducing resistance occur throughout history in many cultures. Perhaps the best known (but by no means the first) is the account of Edward Jenner, who defended the practice of smallpox vaccination. It is now evident that immunity is mediated by specialized organs, cells, and molecules, which together produce the phenomena described by these early observers. 2. The basis for specific immunity is the recognition, at a sub-molecular level, of subtle structural differences between "self" and "non-self". It is this ability to distinguish small molecular differences that makes the molecules and cells of the immune system good candidates for use as specific molecular probes in research, therapeutics, diagnostics, and industry.

C. AN EXPERIMENT CHARACTERIZING ADAPTIVE IMMUNE RESPONSES 1. The adaptive immune system is characterized by four major properties: inducibility, specificity, memory, and non-responsiveness to self. These properties reflect the cellular and molecular events that comprise the immune response. Your own polio vaccination serves as an example to demonstrate these properties: a. INDUCIBILITY Inducibility is inferred from the observation that immunity is not a constitutive phenomenon: i.e.; exposure to an immunogenic agent, or antigen, must occur before immunity is expressed. b. SPECIFICITY Since immunization with one substance does not provide immunity to another, specificity is inherent in the immune response. This property is one reason that the immune response is so well suited to the production of specific tests and tools for the detection of small molecular differences. c. "MEMORY" The property of memory may be deduced from the observation that subsequent to initial antigen exposure, evidence of immune activity is more rapidly detectable upon reexposure. d. NON-RESPONSIVENESS TO SELF Non-responsiveness to self, one of the most important yet puzzling properties of the immune system, is clearly requisite for a system designed to act against substances by recognizing their molecular structures. Obviously, if such a system were reactive to molecular structures of the host, serious injury or death might result. Indeed, the serious clinical symptoms of diseases characterized by immune reactivity to one's own molecular constituents (autoimmune diseases) are evidence of this. How the immune system manages to distinguish "self" from "non-self" remains one of the major problems in immunology.

I. INDUCTION AND EFFECTOR PHASES OF ADAPTIVE IMMUNE RESPONSES A. An immune response may be operationally divided into two phases: 1. The induction phase. This involves specific interactions between structures on the antigen molecule and receptors on the cells that mediate immunity. These interactions stimulate the cells to differentiate and divide. This antigen-induced activation of cells is the first step in an immune response. 2. The effector phase. This entails direct interaction of the immune system's molecules and cells with an antigen, as well as the activation of other cellular and molecular systems, which collectively serve to destroy and remove the antigen. II. 'HUMORAL' vs 'CELL MEDIATED' ADAPTIVE IMMUNE RESPONSES A. The immune system acts via two major effector mechanisms. 1. The humoral immune response is characterized by the appearance of soluble molecules, called antibodies, in the serum (the fluid constituent of blood) of the immunized individual. Antibodies are proteins that interact specifically with structural features on the surface of the antigen. 2. Cell mediated immunity results in the activation of cells that do not secrete soluble antigen-specific mediators of immunity, but instead directly lyse other cells expressing foreign materials upon their surface. B. In addition, the immune system contains a large regulatory component consisting of cells, molecules and receptors, which serve to positively or negatively regulate the activities of these two major effector arms of the response.

III. KINETICS OF IMMUNE RESPONSES A. IMMUNE RESPONSES FOLLOW CHARACTERISTIC KINETICS FIG. 1

Immune responses follow characteristic kinetics. After the first, or primary, exposure to antigen, several days pass before effector activity (either antibody or cell mediated immunity) can be detected. Following this, there is a steady rise, plateau, and subsequent fall in specific immune activity. Upon secondary exposure, however, several differences are noted. First, the lag period is considerably shorter. Second, the activity rises at a more rapid rate and reaches higher levels than in the primary response.

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B. COMPARE AND CONTRAST PRIMARY vs SECONDARY RESPONSES The differences between the primary and secondary response have several implications for immunity, as well as for any attempt to manipulate or exploit the immune system's capabilities or components. 1. The primary response, because of its slower rise time, is rarely able to prevent disease if the offending antigen is pathogenic. The primary response may, however, help in the recovery from disease and the restriction of diseaseproducing materials to a particular site. 2. The secondary response will frequently prevent any overt symptoms of disease, because the offending agent is rendered innocuous by the immune system's effector functions before these symptoms are apparent. This is the basis for vaccination - the deliberate exposure of individuals to nonpathogenic forms of materials that usually cause disease. This intentional exposure 'primes' the subject, so that subsequent exposure will induce a secondary, diseasepreventing response. C. WHAT CELLULAR AND MOLECULAR EVENTS UNDERLIE THIS? These kinetic differences between primary and secondary responses reflect a preferential expansion and differentiation of the cells within the immune system that are specifically reactive with the immunizing anti- gen. Most of these changes occur during the primary response. Thus, upon secondary exposure this already expanded, specific armamentarium reaches detectable levels of activity more rapidly.

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LECTURE 2 SESSION I ADVANCE ORGANIZER: An understanding of the antibody molecule and antibody-antigen interactions shows how specificity can be explained at a molecular level. The precipitin reaction (see below) may be used to demonstrate the properties of antibody-antigen interactions and make inferences about the general structure of antibodies. The major take-home message from the precipitin reaction experiment is that humoral immune responses are inducible, specific. The major point of hapten inhibition of precipitation is that antibody-antigen interactions are reversible and that antibodies are multivalent. Immunoglobulins (antibodies) are large multi-chain glycoproteins. Each molecule has two identical heavy and two identical light chains. The amino terminal portions of both types of chains are highly variable in amino acid sequence, and are where the combining site for antigen is formed. The carboxy-terminal portions of each chain are fairly constant from molecule to molecule and dictate effector functions. Several general classes of heavy and light chain constant regions exist, and define the heavy and light chain "isotypes".

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I. THE MOLECULAR BASIS FOR SPECIFICITY IN HUMORAL RESPONSES A. THE ANTIBODY MOLECULE Understanding the chemical nature of antibodies and their interactions with antigen is important for several reasons. First, their structure demonstrates how exquisite specificity may be expressed at a molecular level. Second, they provide a conceptual framework for more general considerations of molecular recognition in solution and at cell surfaces. B. EXPERIMENTAL APPROACH TO UNDERSTANDING Ab STRUCTURE: 1. PRECIPITIN REACTION Antigen-antibody interactions may be demonstrated and studied by a variety of tests. Detailed descriptions of all such tests are beyond the scope of this course. The following discussion presents the fundamental properties of antibody-antigen interactions using an elegantly simple test, the precipitin reaction. The results of this test make many inferences about the nature of the antibody molecule's structure and chemical behavior. C. IMMUNIZATION - PRACTICAL ISSUES DEFINITIONS ANTIGEN - Any substance that will induce a specific immune response when administered to an immunocompetent individual. HAPTEN - A substance, frequently a small organic group, which cannot itself act as an antigen , but which will serve as an antigenic determinant when coupled to a larger, "carrier", molecule. [e.g.; in the antigen, DNP-BSA (dinitrophenyl - bovine serum albumin), the DNP group is the hapten and the BSA is the carrier molecule. The entire complex is the antigen.] CARRIER - A substance, usually a large molecular weight protein, to which small groups (haptens) may be coupled in order to make them antigenic. ANTIGENIC DETERMINANT or EPITOPE- Any small region of a molecule which forms a 3-dimensional array of structure and charge which will be recognized and bound by an antibody combining site. Note that unlike the above entities, this term has no meaning except when spoken of in the context of a particular antibodys combining site.

D. THAT ANTIBODY CAN CAUSE PRECIPITATION OF ANTIGEN SHOWS: INDUCIBILITY

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There is no precipitation formed in the pre-immune serum sample, only in the samples after immunization. SPECIFICITY As the molecular vehicle of humoral immunity, it stands to reason that the interaction of an antibody with antigen displays specificity. (This specificity is a function of the amino acid composition within particular areas of the antibody molecule, as seen below. The combining site thus formed presents a particular spatial array of structure and charge which is available to interact with small structures on the antigen surface.) In the precipitin test we are doing, the specificity is shown by a lack of reactivity against materials which were not used to immunize. HETEROGENEITY This is actually more a property of the humoral immune response in general than of antibodyantigen interactions per se. In general, most antigens elicit a large variety of antibodies when used to immunize an individual. For example, even very small organic molecules such as 2-,4dinitrophenyl (DNP), if coupled to an appropriate carrier molecule, will elicit literally thousands of different antibodies - each of which binds DNP with measurable affinity but each of which is slightly different than the other. This extensive HETEROGENEITY allows a great deal of selectivity within immune responses, since it is thought that those antibodies of higher affinity will gradually predominate as antigen levels decrease within the individual. Indeed, a gradual increase in the average affinity of an antisera is observed as the primary response proceeds and presumably reflects this process. Heterogeneity is shown in our example by the reaction of the antisera with the hapten - even on a carrier which we know there are no antibodies against, as well as with the carrier molecule we used in the immunization - even when no hapten molecules are coupled to it. E. HAPTEN INHIBITION SHOWS: REVERSIBILITY Perhaps the most important property (at least in terms of your understanding what the term specificity means in an immunological sense) of antibody-antigen interactions is their reversibility. This property is often ignored by veteran as well as novice immunobiologists and is frequently rediscovered by experience. Reversibility of antibody-antigen interactions may be demonstrated by various types of competitive inhibition experiments. The reversibility of antibody-antigen interactions indicates that the binding of an antibody to its ligand is a non-covalent process mediated primarily by charge interactions and other weak chemical forces. This in turn indicates that antigenantibody interactions are an equilibrium process, and may be described (as any other equilibrium process) by the law of mass action. Thus, for any particular antibody-antigen combination, it should be possible to derive an affinity constant based upon the strength of the interaction between the combining site and the ligand. MULTIVALENCY REQUISITE FOR PRECIPITATION

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Since monovalent ligand will competitively inhibit the precipitation of complexes that occurs when multivalent ligands are used, it may be inferred that precipitation requires multivalent interactions, and more importantly, that antibody molecules are likely to have more than one combining site per molecule.

IMMUNOGLOBULIN STRUCTURE I. GENERAL FEATURES All immunoglobulins consist of at least one basic four-chain monomer. Each monomer contains two identical large, or heavy, chains; and two identical small, or light, chains. The heavy chains have a molecular weight of 50K daltons, and the light chains have a molecular weight of 25K daltons. Thus, each such four-chain molecule has a weight of about 150Kd. The chains are linked by interchain disulfide bonds as shown in Figure 2. In addition, each chain has several intrachain disulfide bonds which occur at regular intervals and cause the molecule to fold in distinct globular domains of about 110 amino acids each. FIG. 2 (DRAW AND LABEL AN IG MOLECULE IN THIS SPACE)

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II. PRIMARY STRUCTURE The antigenic specificity of a given immunoglobulin molecule is determined by the aminoterminal 110 amino acids of both chains. In this region of both heavy and light chains, the primary amino acid sequence differs markedly from molecule to molecule. This 110 amino acid long region of each chain is called the variable region. In addition, three areas of particularly great variability exist within this 110 amino acid long variable region, which are termed hypervariable regions. These hypervariable areas contain the amino acid residues actually involved in forming the antigen combining site once the immunoglobulin molecule has folded. (DRAW AND LABEL
A VARIABILITY PLOT IN THIS SPACE)

FIG. 3

The carboxy terminal 110 amino acids of the light chains, and 330 amino acids of the heavy chains, are relatively constant from molecule to molecule. However, there are several different possible constant regions which may be used for each. These different constant regions are termed light- and heavy-chain isotypes. Among light chains, two major types of constant regions are found. Among heavy chains, at least eight types of constant regions may exist. The constant regions of the heavy chains dictate the effector function of the immunoglobulin molecule.

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I. HEAVY AND LIGHT CHAIN ISOTYPES The two types of light chain constant regions are termed kappa () and lambda (). These are the light chain isotypes. The average proportion of antibodies bearing kappa vs. lambda light chains varies greatly between species. For example, in mice over 95% of all antibodies bear kappa light chains, whereas in humans the proportions of kappa and lambda are roughly equal. No functional distinctions have been ascribed to the different light chain isotypes. It is the constant region of the heavy chain that is used to characterize a particular antibody molecule as IgM, IgG1, IgA, etc. These are termed heavy chain isotypes. When used in reference to a complete molecule, the Roman alphabet symbols shown above are used. When reference is made to the heavy chain alone, the Greek alphabet counterpart is generally used (e.g.; gamma, mu, alpha, etc.). Particular quaternary structures are associated with particular heavy chain isotypes. Most isotypes consist of only the single four-chain monomer already described. Notable exceptions to this are IgM and, under certain conditions, IgA. The IgM molecule is a pentamer of the four-chain subunit, held together by short peptides termed the J-chain. Thus, each IgM molecule contains ten antigen combining sites. IgA molecules are initially produced as a monomer. However, this isotype is capable of being secreted across mucosal membranes. During this process, two monomers are joined to form a dimeric, tetravalent molecule.

The potential effector functions mediated by an immunoglobulin molecule are dictated by the heavy chain isotype. This is because many effector functions involve the interaction of antigenantibody complexes with other cells or molecules, and the sites of these interactions are created by the tertiary and quaternary structure of the constant region domains. Thus, constant regions of different primary sequence will differ with respect to these sites.

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Heavy chain constant region isotype

When in a whole molecule, the Ig is termed: IgM( or )

Major functional properties : First isotype made in primary responses (pentameric form), good complement fixation; Surface receptor for antigen on B- lymphocytes (monomeric form). Surface receptor for antigen on mature primary Blymphocytes. Not secreted. These IgG subclasses are the major isotype found in the serum during secondary responses and late primary responses. These are the isotypes generally associated with protection to pathogens, except for certain bacteria, or for pathogens that enter via mucosal routes Associated with mucosal sites (e.g.; peyers patches); Transported across basement membranes Binds to receptors on mast cells and mediates immediate hypersensitivity and anaphylaxis (types of allergy).

1 2a 2b 3

IgD( or ) IgG1( or ) IgG2a( or ) IgG2b( or ) IgG3( or ) IgA( or )

IgE( or )

II. ENZYME DIGESTION: Fab AND F(ab)2 FRAGMENTS Several techniques which fragment immunoglobulin molecules are useful for both the study and the use of antibodies. These techniques produce predictable breaks in immunoglobulin molecules and may be used to alter the valency, in terms of antigen combining sites, as well as to separate the combining sites (amino terminal portions) from the areas of the molecule which mediate effector functions (carboxy terminal portions). The most commonly used technique is enzymatic cleavage. By far the most widely used enzymes for fragmenting immunoglobulins are papain and pepsin. Each of these results in the cleavage of the molecule such that the amino terminal, antigen-binding end of the molecule is separated from the carboxy terminal regions of the heavy chains. Their cleavage points differ: pepsin retains the two combining sites in their usual dimeric configuration; whereas papain separates the two amino terminal arms - resulting in two monovalent antigen binding fragments. The antigen binding fragments produced are termed either F(ab) or F(ab)2; for the monovalent and bivalent fragments respectively. The heavy chain constant fragment that results from such cleavage is called the Fc fragment. These techniques are useful in situations where the effector functions of a molecule hinder the intended use. For example, some constant regions tend to stick to cell surfaces non-specifically, and will thus cause problems if one is trying to identify particular cell types using antibodies specific for particular cell surface structures. In these cases, the use of F(ab) or F(ab)2 fragments will frequently prevent false positives due to constant region binding to cells. III. AFFINITY CONSIDERATIONS AND MEASUREMENT By measuring the proportion of sites filled at given molar concentrations of antibody and antigen, the calculation of affinity constants is possible. The affinity constants of antibodies 18

may vary over a wide range: from 104M-1 to as great as 108M-1. It is not essential that you know the appropriate equations and manipulations needed to derive an affinity constant. HOWEVER, THE CONCEPT OF HOW AFFINITY RELATES TO THE NOTION OF IMMUNOLOGIC SPECIFICITY IS VERY IMPORTANT.

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SESSION 2, ADVANCE ORGANIZER Although antibodies show exquisite specificity for a particular antigen or determinant, they must also share many functions - particularly those that are designed to rid the body of the offending agent. These shared functions are collectively termed effector functions. The constant regions of immunoglobulins dictate their effector functions, and different isotypes mediate different effector mechanisms. Some effector mechanisms are the direct effect of antibody interaction with antigen, such as NEUTRALIZATION. Other effector functions, although initiated by antigen-antibody interaction, require other cells or molecules in order to proceed. Examples of this are OPSONIZATION and COMPLEMENT FIXATION.

THE VARIOUS FUNCTIONS OF THE IMMUNOGLOBULIN ISOTYPES

Heavy chain constant region isotype When in a whole molecule, the Ig is termed: IgM( or )

Major functional properties : First isotype made in primary responses (pentameric form), good complement fixation; Surface receptor for antigen on B- lymphocytes (monomeric form). Surface receptor for antigen on mature primary Blymphocytes. Not secreted. These IgG subclasses are the major isotype found in the serum during secondary responses and late primary responses. These are the isotypes generally associated with protection to pathogens, except for certain bacteria, or for pathogens that enter via mucosal routes Associated with mucosal sites (e.g.; peyers patches); Transported across basement membranes Binds to receptors on mast cells and mediates immediate hypersensitivity and anaphylaxis (types of allergy).

1 2a 2b 3

IgD( or ) IgG1( or ) IgG2a( or ) IgG2b( or ) IgG3( or ) IgA( or )

IgE( or )

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I. ANTIBODIES SHARE EFFECTOR FUNCTIONS One generally assumes that the raison d'etre for the immune system is to detect foreign substances and then rid the organism of these substances. We've seen that the "detection" of these foreign substances is mediated by molecules of exquisite specificity - each molecule having a unique set of antigenic determinants with which it reacts. In contrast, it would be inefficient to be equally specific in the mechanisms of destroying and removing foreign materials, as this would require thousands of different mechanisms where one or a few might suffice. Indeed, most of the mechanisms that serve this purpose are broadly shared. These shared functions are called EFFECTOR MECHANISMS. MOST EFFECTOR MECHANISMS REQUIRE ANTIGEN-ANTIBODY BINDING TO BE ACTIVATED OR TO PROCEED. Humoral effector functions are generally activated or mediated via immunoglobulin HEAVY CHAIN CONSTANT REGIONS. Thus, all antibodies with a particular heavy chain constant region will share the ability to induce a particular effector function, regardless of antigen specificity. Obviously, since several different constant regions exist (the isotypes, remember?); the ability to mediate a particular effector mechanism will differ among the isotypes. II. SOME EFFECTOR MECHANISMS ARE A RESULT OF ANTIBODY BINDING ANTIGEN. THIS IS TERMED NEUTRALIZATION A. This process is particularly important in terms of toxins and viral infection. 1. NEUTRALIZATION OF TOXINS a. Substances that cause severe pathology by binding to receptors and interfering with their normal activities (e.g.; tetanus toxin). b. These may be neutralized if an antibody can bind and prevent the toxin from interacting with its target - this is usually simply a steric hindrance of the toxin's combining site for its target.

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2. NEUTRALIZATION OF VIRUSES a. The infection of cells with viruses can be prevented by antibody binding to the virus. Bound antibody may impede the virus by preventing any of the steps requisite for successful viral infection: 1. adsorption 2. penetration 3. "uncoating" 4. reduction of infectious units III. SOME EFFECTOR MECHANISMS INVOLVE MAKING THE NORMAL PHAGOCYTIC PROCESSES OF MACROPHAGES AND NEUTROPHILS MORE EFFICIENT: OPSONIZATION A. Anything that makes it easier for a phagocyte to ingest material is termed an OPSONIN (From the Greek opsonien - to prepare for eating). 1. Antibodies may act as opsonins in two ways: a. Macrophages and neutrophils have receptors for some constant regions. i. Increase avidity of interaction of the macrophage/neutrophil surface with the antibodycoated substance. ii. High affinity receptors exist for several IgG subclasses (IgG2a is best in mouse, IgG1 in human although there are reports of receptors for nearly all isotypes - but some are remarkably low affinity and their in vivo significance is unclear). b. The binding of antibody of any isotype will tend to neutralize the local surface charge especially for surfaces which are otherwise very highly charged. Since macrophages do not phagocytose highly charged substances as well as neutral ones, this will result in making the material more easily ingested, whether receptors exist for the C-region or not.

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IV. THE IgA ISOTYPE PROVIDES FOR IMMUNE ACTIVITY AT PARTICULAR ANATOMIC SITES: THE SECRETORY IMMUNE SYSTEM A. IgA may be transported across basement membranes 1. Results in antibody in secreted fluids and is thus particularly important at mucosal surfaces: a. gut - may restrict pathogens to gut, prevent systemic disease. Some pathogens can set up local infection but not produce disease, e.g.; polio. b. lung mucosal surfaces - of great importance in protection from pulmonary infection, as well as restriction to portal of entry. c. milk - role in neonatal protection debated, but possible. May also induce proliferation of particular clonotypes through idiotypic interactions, but this is also debated.

2. Mediated by IgA constant region site

3. Valuable in local immunity and restriction of infections to portal of entry. THE COMPLEMENT SYSTEM The complement system is a "cascade" of serum proteins which, when activated by antibodyantigen complexes, serves to lyse cells. This is accomplished by enzymatic activity that results in the creation of non-selective ion channels (i.e.; a hole) in the cell surface. Only certain isotypes can mediate complement fixation. Two major pathways of complement activation exist, termed the Classical pathway and the Alternate pathway. The complement system is tightly regulated by a complex system of positive and negative feedback loops, and interdigitates with other enzyme pathways important to inflammatory processes. The complement system will be discussed in greater detail in the Innate immunity sections and lectures.

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LECTURE 3 SESSION I, ANATOMY OF THE IMMUNE SYSTEM ADVANCE ORGANIZER

The immune system consists of a variety of specialized organs and vessels that facilitate the "surveillance" role of the immune system. These organs are divided into PRIMARY and SECONDARY LYMPHOID ORGANS. Primary lymphoid organs are where lymphocytes are produced. Secondary lymphoid organs are areas to which lymphocytes migrate after they are produced. Lymphocytes are produced constantly, and recirculate at a relatively rapid rate. Most lymphocytes are relatively longlived, in the range of months to probably years, but most myeloid cells are rather short-lived. Most white blood cells recirculate continuously throughout most of their lifetime, though these circulation patterns may be altered when exposed to foreign antigens. The basic recirculation pattern is: blood-to-lymph-to-blood. However upon infection, antigen specific lymphocytes become engaged in peripheral lymphoid organs (lymph nodes, spleen, etc) where they become activated and induced to become effector cells. These effector cells, as well as many innate immune cells such as granulocytes and basophils, can be recruited to sites of infection (eg.: in the skin) via the general process of inflammation. Inflammation at infected sites cause macrophages in these tissues to secrete soluble molecules called cytokines. Cytokines are soluble proteins produced by one cell type that acts on other cells of the immune system. The cytokines produced by tissue macrophages act on local blood vessels, causing them to display ligands that bind to while blood cells in the blood, causing blood leukocytes to bind to and migrate through blood vessels to infected sites. Adaptive immune responses primarily involve three types of cells: B-lymphocytes, T-lymphocytes, and antigen-presenting cells (APCs). By contrast, innate immune responses are chiefly the job of various cells of the myeloid lineage. All arise from hematopoietic stem cells in the bone marrow. B cells are the cells which produce antibody. T-cells mature in the thymus, and may differentiate into one of at least three main subtypes of cells: Cytotoxic T-cells, which can lyse other cells of the body expressing foreign surface molecules; Helper T-lymphocytes, which are involved in the induction of both humoral and cell mediated immunity; and Regulatory T-lymphocytes, which suppress immune responses. APCs may be of several types, although most are dendritic cell-like, and also mature in the bone marrow. These are important in the induction of immune responses through the presentation of antigen to helper T-cells.

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I. PRIMARY VERSUS SECONDARY LYMPHOID ORGANS PRIMARY: MARROW THYMUS SECONDARY LYMPH NODES SPLEEN PEYERS PATCHES GALT/BALT II. RECIRCULATION AND TURNOVER Most mature lymphocytes constantly recirculate through a system of vessels and organs called the reticuloendothelial system. This system includes the lymphatics, lymph nodes, and spleen. In contrast to the thymus and marrow, where lymphocytes are produced, these areas of lymphocyte recirculation and migration are called secondary lymphoid organs. The pattern of recirculation tends to be from blood to lymphatics to blood. The major areas of antigen contact and lymphocyte activation are the lymph nodes, although this may occur in the spleen and elsewhere as well. Almost all white blood cells recirculate, although potentially at different rates. Further, the relative proportion of T- versus B- lymphocytes in these secondary lymphoid organs varies. The lymph nodes tend to be more rich in T-lymphocytes whereas the spleen has a larger proportion of B-lymphocytes. II. CELLS OF THE IMMUNE SYSTEM A. CELLS AND THEIR GENERAL FUNCTIONS All manifestations of immunity arise from cells within the individual. The major cell types involved in the immune response are lymphocytes and macrophages. The lymphocytes are subdivided according to their function and their site of origin. B. LYMPHOCYTES: 1. ORIGIN OF LYMPHOID CELLS All blood cells including lymphocytes originate from pluripotential stem cells in the bone marrow. As these stem cells differentiate along the lymphocytic lineage, they may either mature in the bone marrow, or they may migrate to and mature in the thymus. Because the marrow and thymus are sites of lymphocyte production, they are termed primary lymphoid organs. Lymphocytes are constantly produced throughout the lifetime of an individual. Since there is a steady state of lymphocyte numbers within the body, it is clear that turnover occurs within the mature lymphocyte pool. The precise half-life of unstimulated, or primary, lymphocytes ranges from two weeks to two months or more. If stimulated by antigen, however, a lymphocyte and its clonal progeny may be very long lived - on the order of many years.

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2. T- AND B- LYMPHOCYTES Lymphocytes that mature in the bone marrow are known as B-lymphocytes, and those that mature in the thymus are called T-lymphocytes. The functions of the lymphocytes that develop in these distinct sites are quite different. 3. B-CELLS MAKE ANTIBODY B-lymphocytes are the effectors of humoral immunity, i.e.; they produce antibody. Initially, antibody is produced and displayed as a membrane-bound surface molecule, and it is only after the antigen-driven activation of B-lymphocytes that the antibody is secreted. The membranebound form of antibody acts as an antigen-specific receptor for B-cell activation. 4. T-CELLS PERFORM VARIETY OF TASKS T-CELL SUBSETS T-lymphocytes may serve a variety of functions and are further subdivided accordingly. Three major subsets of T-lymphocytes exist. a. CYTOTOXIC These T-lymphocytes are the effectors of cell mediated immunity. When activated appropriately, T-lymphocytes of this subset can directly lyse other cells displaying foreign molecules on their surface. These cells are called cytotoxic T-lymphocytes, abbreviated CTL. Just as Bcells, CTL also display surface receptors for antigen on their surface, but these receptors are not antibody and they are not secreted in detectable quantity following activation. b. REGULATORY T-CELL SUBSETS The remaining subsets of T-lymphocytes regulate both the humoral and cellular arms of the immune response. Again, each of these cell types bear surface receptors which impart antigen specificity, but these receptors are not antibody and are not secreted upon activation. i. HELPER This regulatory subset of T-lymphocytes is required for the induction of most immune responses. These T-lymphocytes thus "help" immune responses and are accordingly called helper T-lymphocytes, abbreviated Th. ii. Regulatory (suppressor) T cells The second set of regulatory T-lymphocytes acts to down-regulate (suppress) immune responses. 5. DENDRITIC CELLS and MACROPHAGES Macrophages also arise in the bone marrow from pluripotential stem cells. Unlike lymphocytes, macrophages (and all innate immune cells) bear no endogenously synthesized receptor 26

for specific antigen. Instead, the macrophage is able to phagocytose and pinocytose particulate or soluble materials in a relatively indiscriminate fashion. Macrophages and dendritic cells are of great importance throughout the immune response, and serve two major functions: a. ANTIGEN PRESENTATION Both macrophages and dendritic cells are able to "present" antigen to the lymphocytes (especially Th) in a fashion that leads to lymphocyte activation. In addition, macrophages secrete a variety of soluble mediators of lymphocyte activation. (These two properties are discussed in greater detail later). In particular, the follicular dendritic cells of the spleen are effective at presenting antigen during primary responses. b. PHAGOCYTOSIS Second, macrophages also act during the effector phase of the response by ingesting and destroying foreign materials, and are especially effective at ingesting materials coated with antibody. 6. SUMMARY OF MYELOID CELLS Neutrophil: Phagocytic white blood cell (leukocyte) that enters infected tissues in large numbers and engulfs and kills extracellular pathogens. Neutrophils are a type of granulocyte and contain granules that stain with neutral dyes; hence their name. The most abundant white blood cell. Monocyte: Leukocyte with a bean-shaped nucleus. Monocytes are precursors for tissue macrophages. Macrophage: Large mononuclear phagocytic cell found in most tissues. Derived from blood monocytes, macrophages contribute to innate immunity and early nonadaptive phases of host defense by acting as professional antigen-presenting cells and as phagocytes. Basophil: white blood cell present in small numbers in the blood; it is one of the three types of granulocyte. Basophils contain granules that stain with basic dyes. Eosinophil: Leukocyte that is one of the three types of granulocyte. It contains granules that stain with eosin (hence their name) and whose contents are secreted when the cell is stimulated. Eosinophils contribute chiefly to defense against parasites. Dendritic cell: Highly functional antigen presenting cells with a branched, dendritic morphology. They are the most potent stimulators of T-cell responses. Immature dendritic cells take up and process antigens but cannot yet stimulate T cells. Mature or activated dendritic cells are present in secondary lymphoid tissues and are able to stimulate T cells. 7. THE CONCEPT OF SURFACE MARKERS Its hard to tell mononuclear cells apart morphologically. Antibodies against cell surface molecules unique to particular cell types are a useful way to identify, separate, and study otherwise indistinguishable cells. Molecules recognized by such reagents are called cell surface 27

"markers [also differentiation antigens or surface antigens]. Many markers are defined which distinguish functional categories of lymphocytes. CLUSTER DESIGNATION (CD) NOMENCLATURE After it became common to use markers to distinguish cell types, a single nomenclature was decided upon (but not until the existing nomenclature was completely unintelligible, and if you read the old literature, you will find this out). The current nomenclature involves cluster designations - the idea being that particular clusters of markers can distinguish subsets of cells. Thus, most markers are preceded by CD- followed by a number. For example, a marker that is found an all T-cells happens to be called CD3. I could thus say that if the cell is CD3 positive (CD3+), it must be a T-cell.

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LECTURE 4 SESSION #1: THE CELLULAR BASIS FOR ANTIBODY SPECIFICITY ADVANCE ORGANIZER: Clearly cells lie at the foundation of the mechanisms governing all immune responses. A unifying hypothesis to explain the cellular basis of the immune response is the clonal selection hypothesis of Burnet. This hypothesis is generally accepted and serves as a good framework within which to view the processes of immunity at a cellular level. The major tenet of this hypothesis is that specificities (receptors) are clonally distributed. If specificities are clonally distributed, the breadth of antigens to which we can respond demands that the diversity of clonal specificities be enormous. The degree of diversity among B-cells has been estimated at greater than ten million. Although this explains the great specificity and heterogeneity of responses, it raises an intriguing genetic question: How can so many different specificities (in the case of B-cells, antibody variable regions) be encoded in the genome? The answer to this question came from an in-depth analysis of antibody genes.

THE CELLULAR BASIS FOR SPECIFICITY I. NOTION OF RECEPTOR DIVERSITY It follows from our notions about the molecular and cellular bases for specificity that the array of receptors must be large. Otherwise, the immune system might fail to respond to many antigens. This perplexing problem of receptor diversity was studied extensively during the past decade; especially with respect to B-lymphocytes, where the chemical nature of the receptor was known. Indeed, the array of antibody specificities is very diverse. It is estimated that within a species, between ten- and one hundred-million possible combinations of heavy and light chain variable region pairs may be produced, with particular individuals expressing several million within their B-lymphocyte pool at any given time. II. CLONAL DISTRIBUTION OF RECEPTORS A. Specificity is achieved at the cellular level through the clonal distribution of antigenspecific receptors Current thought about the cellular basis of immune responses is based upon the clonal selection hypothesis of Burnet. In general, this hypothesis explains the specificity of immune responses at the cellular level by requiring that the receptors for antigen be clonally distributed, and that the cells of the immune system become activated only following the interaction of these receptors with their corresponding antigen. Although originally formulated to explain the humoral response (i.e.; B-cell responses), the postulates are equally applicable to T-cell responses. 29

III. CLONAL SELECTION HYPOTHESIS A. Lymphocytes express a receptor for antigen on their surface, and interaction of this receptor with ligand (antigen) is requisite for activation of the cell. Further, the specificity of the effector function (antibody specificity, or cytotoxic T-cell specificity) will be the same as the receptor's specificity. (RECEPTOR=PRODUCT) B. At any time, a given cell expresses only one type of receptor (in terms of antigen specificity). Thus, each clone of lymphocytes is monospecific. (MONOSPECIFICITY) C. Following activation, a cell and its clonal progeny will maintain the specificity originally expressed in receptor form. (CLONAL INTEGRITY) A bit of reasoning will show that these tenets will account for the specificity and inducibility of the immune response at the cellular level. In addition, experimental evidence supports the tenets of this hypothesis to the extent that it is accepted nearly axiomatically. D. It is now evident that antigen-specific receptors on B-cells are antibody molecules, and the specificity of the secreted antibody product of each B-cell clone is equivalent to its receptor. Although changes in specificity may occur within a clone of activated B-cells by point mutation within the genes that encode the antibody, the notion of clonal integrity is still generally valid. E. T-cell receptors are clonally distributed as well, although a detailed discussion of this will wait until we have discussed certain aspects of T-Cell antigen recognition by Dr. Koretsky. IV. RECEPTOR DIVERSITY A. LIMITING DILUTION ANALYSES demonstrate enormous diversity in the repertoire of clonally distributed B-cell and T-cell receptors.

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LECTURE 4 SESSION II, ADVANCE ORGANIZER MOLECULAR GENETICS OF ANTIBODIES Given this great degree of diversity, several intriguing biological problems were evident: First, how is so diverse an array of molecules maintained or generated in the genome of an individual? Second, how is it genetically possible to conserve the primary sequence of one end of a protein molecule while allowing the other end to vary so greatly? The key to these questions lay in the discovery that the genes encoding a complete heavy or light chain do not occur as contiguous segments of DNA in the germ line genome. Instead, several gene segments, which are separated by considerable distance in the germ line DNA, are spliced together at the DNA level during the differentiation of B-cells. In this manner, many permutations of these different segments may be achieved even with a relatively small number of gene segments.

I. PROTEIN SEQUENCE ANALYSES PROVIDED CLUES TO THE GENETICS OF DIVERSITY A. kappa light chains B. lambda light chains C. germline versus somatic mutation II. THE NOTION OF MULTIPLE GENE SEGMENTS A. A functional light chain gene is encoded in the germ line genome by three segments. One segment encodes the constant region, and there is one copy of each type of light chain constant region per haploid genome. B. Each constant region gene segment is associated with two collections of additional gene segments which can encode the variable region of a light chain protein. These two sets are termed the light chain V-region genes (VL) and J-region (JL) genes. C. There are 4 to 5 JL-region gene segments, each of which may encode the 14 amino acids amino terminal to the constant region. D. There are approximately 100 VL-region genes that can each encode the remaining 96 positions amino-terminal to the J-region. The J-region was so named because it is the segment which "Joins" the V- and C-region genes. The J-region genes are found fairly close (less than 31

2kb) from the C-region genes in the germ line genome. The J-region genes are separated from one another by introns of less than 2Kb as well. The V-region genes, however, are found clustered a considerable distance (at least 17kb) from the J-region genes. E. During differentiation, cells destined to become B-cells rearrange their DNA in this region and cause one of the V-region genes to be brought into association with a J-region gene. The resulting VJ-C combination is then transcribed, post-transcriptionally modified, and translated. The signals for appropriate joining are palindromic sequences found immediately 3' of each J-gene and immediately 5' of each V-gene. It is presently believed that any V- may be arranged with any J-. Thus, for kappa light chains this results in 400 V-J combinations. It is important to recall that the region encoded by the area of the V-J junction is located within one of the hypervariable regions, and will thus be likely to influence antigen specificity. Therefore, from approximately 100 genes over 400 choices of kappa light chain specificities may be derived. A similar set of genes, although apparently not as extensive, appears to exist for lambda light chains. Kappa and Lambda genes are not linked and thus represent independent sets of V, J, and C genes (termed V, J, and C, and V, J, and C). II. Heavy chains are produced by joining four gene segments The strategy for generating diversity among heavy chains is similar to that of light chains, but instead of only two gene segments contributing to the formation of the variable region of the protein, three segments are involved. As among light chains, several gene segments exist just upstream of the heavy-chain constant region genes which encode a joining, or JH, region of the heavy chain. Also, a cluster of VH genes, which encode the amino terminal 96 amino acids of the heavy chain, are found a considerable distance 3' to the JH genes. Unlike light chains, a third group of gene segments exists between the VH and JH region genes. This third type of gene segment used in the formation of the heavy chain variable region is called the D region (for Diversity). Thus, during the formation of a functional heavy chain gene, a VH, D, and JH gene are juxtaposed using palindromic sequences which flank each segment as markers for splice sites, leading again to a large variety of permutations based on a small number of gene segments. In addition, it is not essential that a D gene be used (i.e.; some heavy chains are made up of only a VH-JH join), so a further dimension in diversity generation is gained via the exclusion of D-regions in some heavy chains. It is presently felt that there are about 5 JH, 10 to 20 D, and 300 to 400 VH genes per haploid genome. The arrangement of heavy chain constant region genes also differs from that observed for light chains. Although as in light chain genes, each possible heavy chain constant region gene (which will determine the isotype of the heavy chain produced) is represented once per haploid genome, they are arranged linearly on the same chromosome. Further, the three 110 amino-acid domains of each heavy chain constant region are encoded by exons separated by short introns and for each type of constant region there are two types of introns that can be used for the carboxy-terminal domain. This allows the carboxy-terminal domain to be either a trans-membrane form (which has an appropriately hydrophobic region commensurate with trans-membrane proteins) or a secreted form, depending upon which intron is chosen during post-transcriptional modification of the primary transcript.

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The order of heavy chain constant region genes is generally the same as the order in which the various isotypes are seen during cell differentiation and activation: the gene for C-mu is closest to the JH genes, followed by delta, the various gamma subclasses, alpha, and epsilon. The exact molecular mechanism whereby the genetic rearrangements required for VL-JL or VH-D-JH joining, as well as those required for the switching of heavy chain isotypes during Bcell activation, are not yet understood. IV. DIVERSITY IS GENERATED BY SEVERAL MECHANISMS A. Combinatorial association It is assumed presently that virtually any light chain may be paired with any heavy chain. The multiplication of all possible VLJL combinations by all possible VHDJH combinations shows that nearly 107 antibody specificities may be generated from a collection of about 500 gene segments. B. Junctional diversity In addition to the diversity inherent in the permutations of gene segments available, several additional diversity generating mechanisms exist. The first of these is called junctional diversity. During the rearrangement of VL to JL, and VH to D to JH, some mismatch will occur. As long as frameshift errors are not created, this means that the amino acids encoded in these junctional areas may vary, depending upon the exact codon created. This mechanism of junctional diversity has been clearly confirmed by comparing amino acid sequences of myeloma proteins to the genes available to encode them. C. Somatic variation Further diversity is generated by point mutations which occur during the lifetime of a B-cell clone. It has recently been estimated that the mutation rate in this region may be as high as one mutation per 103 base pairs per generation. This mechanism, assuming the B-cell clone is longlived, could result in a considerable increase in diversity over that afforded by the simple shuffling of germ line gene V, D, and J segments.

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LECTURE 5 SESSION I, THE B CELL RESPONSE B cells function primarily to secrete specific antibodies to potentially dangerous pathogens. Although we've spent some time talking about the cellular and molecular basis for B cell specificity, these models do not explain how the resulting antibodies act to remove or effect the destruction of these pathogens. This lecture will deal with how different types of antigens induce B cells to secrete various antibody isotypes and how these different isotypes elicit one or more of the several unique effector activities of the immune system. We will also discuss the cellular and molecular explanations for affinity maturation, the process whereby antibody affinities for the immunizing antigen INCREASE during the course of a primary humoral response, leading to the generation of high-affinity (and therefore more effective) antibodies. Many of these processes can be explain with an understanding of germinal centers, unique microanatomic structures that form in lymphatic tissues upon antigen-driven B cell activation.

THREE CATEGORIES OF ANTIGENS FOR B CELL RESPONSES T-cell independent type I - B cell mitogens such as lipopolysacharide (LPS). These are not antigens in the sense that they do not bind to antigen-specific receptors on the surface of B cells. In fact, LPS induces B cell proliferation irrespective of the B cell specificity. T-cell independent type II - Antigens that can effectively stimulate antibody secretion in B cells without interacting with T cells. These antigens are typically long, highly repetitive structures that can efficiently cross-link multiple B cell receptors on a given B cell. T cell dependent - tend to be protein antigens to which B cells can bind through the B cell receptor but not effectively respond to without the addition of additional factors derived from T cells. ISOTYPE SWITCHING and AFFINITY MATURATION B cell responses to T-cell independent type II antigens tend to be predominated by IgM and, to a lesser extent, IgG3. In contrast, T-dependent antigens can elicit a wide array of antibody isotypes depending on a variety of factors that we will discuss in greater detail in the second half of the course. In addition, in the 1970's it was first appreciated that antibody affinities for an immunizing antigen increase over time during a primary immune response to a T-dependent antigen. This phenomenon is now thought to be due to two distinct mechanisms, competition between low and high affinity clones (the high affinity ones win!) and a somatic hypermutation mechanism, which introduces random point mutations in the V genes of antigen-reactive B cells. The hypermutation mechanism is thought to be active solely in germinal center B cells.

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Germinal centers are unique anatomic structures enriched for activated B cells. A germinal center can be subdivided into the dark zone, which is highly enriched for proliferative activated B cells undergoing somatic hypermutation, and the light zone which contains postproliferative B cells, follicular dendritic cells (FDCs) that are coated with foreign antigens, and small numbers of helper T cells called Follicular Helper T cells. It is thought that dark zone B cells bearing mutations in their V region genes migrate to the light zone where they compete for access to T cell and FDC-derived signals. Newly mutated B cells bearing high-affinity BCRs (due to somatic hypermutation) are more likely to be selected for, and become long-lived plasma cells or memory B cells. Interactions between B cells and helper T cells. For activated B cells to generate germinal centers and plasma cells in response to T-cell dependent antigens, they must receive a variety of signals from activated helper T cells. These signals take two general forms: cell surface ligands on activated T cells that bind to and activate receptors on B cells, and soluble molecules called cytokines that bind to specific cytokine receptors on B cells. The chief cell surface ligand on activated T cells relevant to antibody responses in called CD40-ligand, or CD40L, which binds to CD40 on B cells. Without CD40 or CD40L, isotype switching and germinal center formation do not occur (this is true for both mice and people). Activated helper T cells also secrete the cytokines IL-2, IL-4, IL-5, and IL-6. IL-2 amplyfies T cell proliferation whereas IL-4 and IL-5 amplify B cell proliferation and class switching, and IL-6 enhances plasma cell differentiation.

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LECTURES 6 & 7 INNATE IMMUNITY I AND II ADVANCE ORGANIZER The immune system employs a variety of cells and physical barriers to protect us from infection. By contrast to adaptive immune functions, these components of the immune system are constitutive and relatively non-specific. Innate immune cells belong to the myeloid lineages of hematopoietic cells. These cells sense tissue damage and pathogens through the action of Toll-like receptors (TLRs) expressed on their cell surface. TLRs bind to structural features or patterns that are defining features for classes of microorganisms. For instance, TLR4 binds to lipopolysaccharide (LPS), a component of the cell wall of all gram-negative bacteria. TLR activation leads to the synthesis of inflammatory cytokines, which in turn recruit additional innate cells to sites of infection, leading to pathogen destruction and activation of key components of the adaptive arm of the immune system. Many innate cells function as phagocytes, and thus can clear infections by ingesting and destroying pathogens. Such infections can also induce complement activation, which amplifies the inflammatory response and enhances phagocytosis by opsonizing pathogens. I. Cells of the innate immune system:

Neutrophils: Phagocytic white blood cell (leukocyte) that enters infected tissues in large numbers and engulfs and kills extracellular pathogens. Neutrophils are a type of granulocyte and contain granules that stain with neutral dyes; hence their name. The most abundant white blood cell. Monocytes: Leukocyte with a bean-shaped nucleus. Monocytes are precursors for tissue macrophages. Macrophages: Large mononuclear phagocytic cell found in most tissues. Derived from blood monocytes, macrophages contribute to innate immunity and early nonadaptive phases of host defense by acting as antigen-presenting cells and as phagocytes. Basophils: white blood cell present in small numbers in the blood; it is one of the three types of granulocyte. Basophils contain granules that stain with basic dyes. Eosinophils: Leukocyte that is one of the three types of granulocyte. It contains granules that stain with eosin (hence their name) and whose contents are secreted when the cell is stimulated. Eosinophils contribute chiefly to defense against parasites. Dendritic cells: Highly functional antigen presenting cells with a branched, dendritic morphology. They are the most potent stimulators of T-cell responses. Immature dendritic cells take up and process antigens but cannot yet stimulate T cells. Mature or activated dendritic cells are present in secondary lymphoid tissues and are able to stimulate T cells.

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Natural Killer cells: These are lymphoid cells (NOT myeloid) that lyse host cells that have become tumor cells or when they are infected with viruses that cause the down-regulation of MHC molecules (more on MHC in the next section). II. Pattern Recognition Receptors

Innate (and some adaptive) immune cells employ Pattern Recognition Receptors (PRRs) to recognize shared molecular patterns associated with certain classes of pathogens. There are four main families of PRRs: 1. 2. 3. 4. Toll-like Receptors (TLRs) C-type lectin Receptors (CLRs) Nod-like receptors RIG-I like receptors

TLRs and CLRs are cell surface receptors, the others are internal to the cell (cytosolic). Each member of these families binds to a particular structural motif associated with a class of pathogens. The main consequence of PRR activation is the induce synthesis of cytokines such as IL-1, IFN, and TNF (see Figure on next page showing the consequences of TLR and NOD activation in immunity). As shown, often these cytokines are the first immune activity in response to infection, and these cytokines can act to initiate and amplify inflammatory responses to these infections. Two key cytokines produced by tissue-resident macrophages in response to infection are IL-1 and TNF. Together IL-1 and TNF enhance the recruitment of other leukocytes (such as neutrophils) from the blood to sites of infection.

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III. Complement The Complement system provides a mechanism to cause the lysis of pathogens, to enhance recruitment of cells it sites of infection (thereby amplifying the inflammatory response), and enhance the phagocytosis of pathogens through Opsonization (binding of Complement fragments to the surface of pathogens, followed by their recognition by receptors on the surface of phagocytes). The Complement system consists of some 30 serum and membrane proteins. Active components of Complement are produced from inactive precursor proteins by a series of proteolytic reactions. There are nine well-known serum proteins in the activation cascade and they are named C1, C2, C3, C9. The basic order of activation during Complement activation is: C1 C4 C2 C3 C5 C6 C7 C8 C9. With inactive complement proteins become active, the resulting protein fragments are labeled by lower case letters (eg. C3 becomes C3a and C3b, C4 becomes C4a and Cdb, etc). Furthermore, C5 C6 C7 C8 and C9 can form a lytic complex that, when bound to a cell surface, creates holes in the cell surface, thus killing the cell in question. 38

There are at least THREE pathways whereby the Complement system can be activated (pictured below). The Classical pathway is initiated by antibodies binding to pathogens. C1 binding to antibody Fc regions causes the degradation and activation of C4 and C2, thus creating C4bC2b complexes that lead to C3 activation (see Figure below). The Alternative pathway is activated by binding of fragments of the complement component C3b to microbial surfaces. The mannose pathway is activated when mannosebinding lectins recognize and bind to foreign polysaccharides on microbial surfaces. This event causes cleavage C4 and C2 in a manner that is similar to the antibodymediated Classical pathway (see Figure below). There are several key negative regulatory mechanisms for the Complement system. Loss of any of these can have dire consequences, leading to aggressive and deleterious inflammatory responses with secondary effects such as autoimmunity and anemia.

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LECTURES 9 & 10 THE MHC AND ANTIGEN PRESENTATION I AND II ADVANCE ORGANIZER Many pathogens, such as viruses, gain entry inside cells where they become invisible to antibodies. Consequently the immune system also employs strategies to identify infected host cells. In sum, peptides derived from pathogen proteins are generated within infected cells, and these are displayed on the cell surface together with specialized host cell surface molecules encoded by a set of genes called the Major Histocompatibility Complex (MHC). Pathogen-specific T cells recognize pathogen-derived peptides complexed with host MHC molecules and respond by generating effector cells including activated helper cells (CD4+) and cytotoxic T cells (CD8+) which act to kill virus infected cells. There are two categories of MHC molecules: MHC Class I molecules and MHC Class II molecules. Class I molecules present peptides to CD8+ T cells, and Class II present peptides to CD4+ helper T cells. Class I molecules are expressed by virtually all cells. In sharp contrast, the expression of Class II molecules, which serve to stimulate helper T cells, is restricted to dendritic cells, macrophages, and B cells. Therefore these cells serve as Antigen Presenting Cells or APCs for CD4+ T cells. Dendritic cells are highly effective APCs.

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I. ANTIGEN PROCESSING AND PRESENTATION Antigen processing and presentation occurs through one of two intracellular trafficking pathways. Endogenous processing pathway - Class I Proteins synthesized within the cell are degraded and the resulting peptides associated with Class I molecules, presumably during transit through the golgi. Exogenous pathway - Class II Proteins ingested from the extracellular environment are digested and processed through the lysosomal system, which results in selective association of resulting peptides with MHC class II molecules. This may result through co-internalization of surface Class Ii molecules during the phagocytic process. Dendritic cells, which express Class I and Class II MHC, initiate immune responses by ingesting pathogens and processing and presenting pathogen-derived peptides to CD4+ and CD8+ T cells. This process involves the ingestion of pathogens or pathogenic material at sites of infection, and migration to a draining lymph node (or the spleen) where dendritic cells present antigens to T cells.

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Peptides derived from pathogens become fixed in a peptide binding grove of MHC Class I or Class II molecules (see Figure immediately below).

II. T CELL RECOGNITION T cells express clonotypic receptors that are similar but distinct from B cells. The vast majority of T cell receptors (TCRs) for antigen are a heterodimer consisting of an alpha and a beta polypeptide. A small fraction of T cells express an alternative TCR composed of a delta and gamma polypeptide. IMPORTANT: T cells recognize a composite structure generated by a foreign peptide and the alpha helices of a self-MHC molecule (see left).

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III. THE MHC LOCUS The MHC locus consists of a large cluster of tightly linked genes that include all Class I and Class II genes. Other genes not related to antigen presentation and called Class III genes collectively. Human MHC genes are called HLA (Human Leukocyte Antigen) genes.

IV. AN UNUSUALLY HIGH DEGREE OF POLYMORPHISM CHARACTERIZES THE CLASS I AND CLASS II GENES OF THE MHC A. EACH LOCUS IN THE MHC HAS MANY ALLELES The degree of polymorphism in the MHC is one of the greatest known in mammalian genetics. The evolutionary reasons for this are debated, but may be a function of the MHC gene product's roles in normal immune responses.

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V. CLASSIC EXPERIMENTS OF ZINKERNAGEL AND DOUGHERTY USING INBRED MICE SHOWED THAT CYTOTOXIC T-CELLS COULD ONLY KILL TARGETS OF THEIR OWN MHC TYPE: A. USED CTL RESPONSE TO VACCINNIA VIRUS TARGETS: Primed CTL: A STRAIN CTL B STRAIN CTL A normal cells of: B vaccinia infected A B + +

This experiment illustrated the phenomenon known as "MHC RESTRICTION:" T cells can only recognize foreign antigens (peptides) when they are presented in the context of a self-MHC molecule. VI. ALLORECOGNITION Due to MHC polymorphisms, when T cells are exposed to APCs from another individual a large fraction of these cells respond by generating activated helper and CTL (killer) cells. THEREFORE, the high degree of polymorphism of MHC genes creates substantial hurdles for transplantation surgeons. Do to the high level of polymorphism in MHC genes, it is exceptionally unlikely that two unrelated individuals with possess the same HLA alleles. Indeed, even close relatives will have numerous unique alleles. [Only IDENTICAL twins will have the same HLA alleles.] Therefore transplantation will always induce the activation of alloreactive T cells.

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LECTURE 11 T CELL DEVELOPMENT AND ACTIVATION There are multiple types of peripheral T cells, each defined by the unique effector activities that they take on in response to infection. All of these cells must be devoid of reactivity for self antigens, but at the same time these cells only see foreign antigens in the context of a self-MHC molecule. How is this possible? Understanding how T cells develop from precursors in the thymus provides important clues to these problems, while also providing a framework for understanding T cell activation by foreign antigens and fully functional antigen presenting cells. I. T cell development in the thymus. Ultimately T cells arise from hematopoietic stem cells in the bone marrow. Poorly characterized lymphoid biased progenitors enter the thymus from the blood where they receive signals that cement their identity as T-lineage cells. At roughly this time TCR gene undergo V(D)J recombination, resulting in the generation of new TCR+ T-lineage cells. As TCRs are first forming on T-lineage cells they also express two surface molecules: CD4 and CD8. Because they express both CD4 and CD8 these cells are often called double positive thymocytes (see Figure). The vast majority of thymocytes are CD4+ CD8+ double positives, and the earliest TCR+ cells are found within this population. New TCR+ double positive thymocytes soon die unless their TCR binds to a self-MHC molecule. This event is called positive selection. T cells that bind Class I MHC molecules become Class I-restricted CD8+ (CD4-) single positive thymocytes, and these cells are destined to become CTLs if activated by antigen. Conversely, T cells that bind Class II MHC molecules become Class IIrestricted CD4+ (CD8-) single positive thymocytes, and these cells are destined to become helper T cells. T cells that fail to bind any self-MHC molecules die via a process called death by neglect! Through positive selection in the thymus, the T cell system defines self. Thus, perturbations in self MHC, through the loading of peptides derived from foreign protein antigens into the peptide binding cleft of an MHC molecule, alert specific T cells of a violation in self, thus providing the first signal leading to T cell activation and initiation of T cell effector activities.

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II. T cell activation by antigen presenting cells. Nave CD4+ and CD8+ T cells remain quiescent throughout their lifetime unless they are at some point activated by foreign antigen. For T cells, foreign antigens are recognized only as peptide fragments displayed in the context of a self-MHC molecule on the surface of an antigen presenting cell (APC). Significantly, signals generated by the TCR in this fashion are necessary for T cell activation, BUT they are NOT sufficient. Additional signals, called co-stimulation signals, are also required. These second or costimulatory signals are provided by activated APCs such as dendritic cells. The chief costimulatory receptor on T cells is CD28, and its main ligands (expressed by mature APCs) are B7.1 and B7.2. In addition, activated T cells are induced to express CD40-ligand (CD40L), and dendritic cells, macrophages, and B cells express CD40 (see Figure).

Dendritic cells and other APCs become fully functional (or mature) when they are exposed to inflammatory cytokines. In this fashion, cytokines generated by innate immune cells serve to activate dendritic cells, which in turn activate Ag-specific T cells. Activated T cells then begin to produce cytokines such as IL-2, and to proliferate. III. The generation of effector helper T cells with distinct cytokine secretion profiles. Upon activation CD4+ helper T cells not only secrete cytokines such as IL-2, they also adopt unique effector lineages defined by different patterns of cytokine expression: TH1 cells secrete IFN, TH2 cells IL-4, and TH17 cells IL-17. In addition, though this process is understood poorly, CD4+ T cells can also become regulatory T cells with immune suppressive activity. As shown in the left-most Figure on next page, each plays distinct roles in immunity to different classes of pathogens. Notably, TH2 cells are associated with antibody responses and surveillance of barriers such as in the gut, and TH1 cells are critical for systemic immunity, owing in part to their role in activating innate immune cells such as macrophages (see rightmost Figure on the next page). This explains why people with defects in CD4+ T cell development or function experience compromised macrophage function.

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LECTURE 12 TOLERANCE ADVANCE ORGANIZER The immune systems development is a complex and intriguing problem. Think about it: The system has to generate several interdigitating systems of receptors; must avoid producing receptors that will react with self while at the same time insuring that it can see enough foreign structures to be protected from disease; must constantly be interacting with the environment and tailoring the existing system to new environmental assaults, and must control the proliferation and activities of the cells involved in immune responses. The driving forces underlying lymphocyte development and tolerance are not 100% clear, although current evidence suggests that several mechanisms are at play. The tolerance issue can be broken down into two distinct conceptual frameworks: central tolerance versus peripheral tolerance. Central tolerance mechanisms operate in central lymphoid organs where lymphocytes are generated. The basic idea here is that self-reactive cells are eliminated soon after they are generated, before they migrate into peripheral lymphoid tissues. Notably, because tolerance induction is an active process, requiring binding of self-antigens to antigen-specific receptors on self-reactive lymphocytes, newly generated lymphocytes must be exposed to the relevant self-antigen while in the thymus (for T cells) or bone marrow (for B cells). By contrast, peripheral tolerance mechanisms operate in peripheral lymphoid tissues, and several distinct mechanisms appear to be essential to maintain tolerance to peripheral self-antigens. It should be emphasized however that central and peripheral tolerance models are not mutually exclusive. Indeed, given the importance of maintaining tolerance to self-antigens, several different processes may be required to avoid autoreactivity. I. CENTRAL TOLERANCE MECHANISMS CLONAL DELETION describes the process whereby self-reactive lymphocytes die (by apoptosis) when they encounter self-antigen. In this way, potentially pathogenic lymphocytes are eliminated from the B or T cell repertoire. Clonal deletion may work chiefly in the context of central tolerance. This viewpoint is supported by data showing that the vast majority of newly formed T and B cells fail to become mature peripheral cells. CLONAL ANERGY describes an alternative process whereby self-reactive lymphocytes are rendered non-responsive to antigen. RECEPTOR EDITING describes another mechanism in which stimulation of self-reactive B cells with antigen induces additional V(D)J recombination events, thus allowing a self-reactive cells to revise its specificity and thus avoid autoreactivity. There is evidence for receptor editing for B cells but not T cells.

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II. PERIPHERAL TOLERANCE MECHANISMS A. THE TWO-SIGNAL MODEL OF T CELL ACTIVATION AND TOLERANCE. This model holds that stimulation of the antigen-specific receptor (signal 1) on a mature T cell without costimulation (signal 2) leads to tolerance rather than activation: SIGNAL 1 + SIGNAL 2 = ACTIVATION SIGNAL 1 ALONE = TOLERANCE Thus, self/non-self discrimination is influenced by the regulation of signal 2 on APCs such as dendritic cells and/or B cells. B. FAS-FAS LIGAND AND PERIPHERAL TOLERANCE This model predicts that signal one results in upregulation of a death receptor termed fas. When fas interacts with its ligand [fas ligand or fasL], a series of intracellular enzymes termed caspases are activated which results in the ultimate degradation of the cellular genome and cell death. This type of cell death is termed apoptosis. Presumably, a costimlation signal (signal 2) will somehow interfere with fas derived signals and thus prevent signal 1 induced apoptosis. C. CTLA-4, A NEGATIVE REGULATOR OF COSTIMULATION CTLA-4 and CD28 are both receptors for B7.1 and B7.2. However, unlike CD28, which sends a positive regulatory signal to the stimulated T cell, CTLA-4 sends a negative signal that dampens the T cell response. The critical importance of CTLA-4 is best illustrated by CTLA-4 knock out mice. These mice display uncontrolled T cell proliferation and die of autoimmunity by 6 weeks of age. D. REGULATORY T CELLS REGULATORY T CELLS suppress immune responses by releasing inhibitory cytokines. These cells are also critical for maintaining tolerance, as induced depletion of regulatory T cells in adult mice causes life-threatening autoimmunity. Regulatory T cells arise from the thymus along with other T cell lineages, but can also be generated de novo from peripheral CD4+ T cells.

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Sample questions
Part I Knowledge Base 1. Which of the following are basic properties of the immune system? A. Specificity B. Inducibility C. Memory D. Non-responsiveness to self E. All of the above 2. Secondary immune responses... A. Display a more rapid onset than primary responses. B. Reach higher maximum levels than primary responses. C. Display a more acute rise (steeper slope) in immune activity over time than primary responses. D. Will often prevent disease E. All of the above. 3. Which statement about antibodies is FALSE? A. They are composed of one or more 150 kd four-chain units. B. They interact with antigens through irreversible, covalent chemical bonds. C. The N-terminal sequences of their heavy and light chains vary from molecule and form the antigen combining sites. D. They are produced by B lymphocytes. E. They have two or more antigen combining sites per molecule. 4. Neutralization.... A. results in the lysis of virus infected cells. B. affords protection from toxins, venoms, and viral infections. C. is usually mediated by secreted IgD. D. is mediated primarily by dendritic cells and macrophages. E. is mediated by mast cells and basophils. 5. Opsonization.... A. is mediated by the interaction of antibodies with the Fc receptors on phagocytes. B. results in more efficient ingestion of antigen by phagocytes. C. is important in resisting and/or fighting many bacterial infections. D. is particularly well-mediated by several of the IgG isotypes. E. all of the above.

to molecule,

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6. Which of the following is not a component of the innate immune system? A. B. C. D. E. Phagocytes Complement NK cells Antigen-experienced memory T cells Epithelial barriers

7. A 6-year-old boy is diagnosed with acute lymphoblastic leukemia. Flow cytometric analysis of his blood cells reveals an abnormal and dominant cell population defined as CD19+ TdT+ and to lack surface Ig expression. Based on these data what is the lineage and developmental stage of these cells? A. B. C. D. Pre-T cell Mature B cell Pre-B cell Memory T cell

8. A patient possessing homozygous null mutations in the gene encoding AID would experience which of the following categories of immunodeficiency? A. Hypo IgM B. Hyper IgM C. Severe combined 9. A patient possessing homozygous null mutations in the gene encoding RAG1 or RAG2 would experience which of the following categories of immunodeficiency? A. Hypo IgM B. Hyper IgM C. Severe combined immunodeficiency due to loss of B and T cells 10. A 34-year-old male experiences an enlarged inguinal lymph node. A biopsy is performed and histological analysis shows that the lymph node architecture is massively disrupted and that the lymph node contains large numbers of cells that are CD20+ TdT- and surface IgG1+ kappa+. A diagnosis of diffuse large cell lymphoma is made. Analysis of the Ig heavy chain genes in these cells shows that one of the two IgH alleles is heavily mutated across the V region. Based on these data what is the most likely origin of these cells? A. B. C. D. The thymic cortex The nave B cell pool A germinal center The bone marrow

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11. Recall that IgM antibodies are the chief heavy chain isotype induced in response to encapsulated bacteria. The effector cell or mechanism most likely to act in concert with these antibodies is: A. B. C. D. ADCC NK cells Complement-mediated Opsonization Cytotoxic T cells

12. The function of the thymus was unknown before 1960. Children with an enlarged thymus were often subjected to irradiation to functionally ablate this organ. These individuals were likely to: A. B. C. D. E. Suffer recurrent viral infections Experience decreased serum IgG titers Experience opportunistic pulmonary fungal infections A & B only All of the above

13. Dendritic cells (DCs) specialize in the endocytosis of pathogens and become activated particularly if their toll-like receptors (TLRs) are engaged by pathogen products. Consider the following three statements about human TLRs: 1) TLRs are thought to bind mammalian self-antigens which are then presented to activated B cells 2) TLRs often bind to different pathogen-associated molecular pattern (PAMP) motifs on pathogenic products. 3) TLR engagement can induce DCs or macrophages to express B7 - a co-stimulatory molecule. The B7 and antigen presentation then occurs to T cells in secondary lymphoid organs, causing the T cells to become activated. Of these statements: A) All are correct B) none is correct C) 1 and 2 are both incorrect D) 2 and 3 are correct. 14. Follicular Dendritic Cells are specialized cells that: A) present antigen only to Th1 cells B) display antigen to B cells during affinity maturation C) are important only for development of innate immune responses D) facilitate endocytosis of blood-borne antigens in the marginal zone

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15. Somatic hypermutation: A) only occurs in B cells B) requires expression of AID C) can lead to affinity maturation D) is one means of increasing antibody diversity E) all of the above are true 16. A virus-infected cell typically presents viral peptides: A) in MHC class II molecules to CD8 T cells B) in MHC class II molecules to NK cells C) in MHC class I molecules to CD8 T cells D) in MHC class II molecules to professional APCs 17. The class II MHC molecule differs from the class I molecule in that: A) it presents peptides to CD4+ T cells B) it is found on a wider range of cell types throughout the body C) it forms a hot-dog bun-like structure, into which the peptide can fit D) it contains b2 microglobulin E) all of the above 18. Myasthenia gravis, characterized by blockade of neuromuscular acetylcholine receptors, is an example of which type of hypersensitivity? A) type I (Immediate) B) type II (Antibody-mediated) C) type III (Immune Complex mediated) D) type IV (T cell mediated) 19. IgE-mediated mast cell degranulation in response to an allergen is an example of which type of hypersensitivity? A) type I (Immediate) B) type II (Antibody-mediated) C) type III (Immune Complex mediated) D) type IV (T cell mediated) 20. Which statement about T-lymphocytes is TRUE? A. They arise from pluripotential bone marrow stem cells. B. They are produced continuously. C. Each one expresses antigen receptors of a single specificity. D. Binding and cross -linking of their antigen receptors is required for activation. E. All of the above statements are true.

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Part II - Analysis/Problem Solving

1. Cytokine receptor levels were measured on T lymphocytes at various intervals following antigen exposure and gave the following results:
HOURS

AFTER ANTIGEN EXPOSURE 24 40 48 100 72 75 96 25 120 5

0 RECEPTOR LEVEL (% of maximum) These results show that: 0

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A. cytokine receptors are inducible. B. cytokine receptors are antigen-specific. C. cytokine receptors bind nominal antigen presented in MHC context. D. cytokine receptors control alloreactivity. Information for questions 2-4: In gene knockout mice, a gene (or set of genes) is eliminated so that the function(s) associated with its product are absent. The table summarizes a flow cytometry analysis of thymic and marrow cells from 5 different knockout mice, named A, B, C, D, and E. A + indicates that cells of the listed phenotype are present, and a - indicates they are absent. Thymus K.O. Mouse A B C D E Marrow

CD4 8 + + + -

+ +

CD4 8 + + -

+ -

CD4 8 + + -

- +

sIgM+ + + + -

Based on these phenotypes and your knowledge of the immune system, which mouse best corresponds to each of the following three gene knockouts? ____2. A m heavy chain gene knockout. ____3. A Class II MHC gene knockout (All class II genes knocked out). ____4. A RAG knockout ( RAG is a protein necessary for all V->D->J gene rearrangement processes)

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Information for questions 5-8: Duane and David are Mom and Dads biological children. You performed MLR and CML assays with all responder/killer - stimulator/target combinations within this family. The assays gave similar results, so they are summarized in the single table below. A + indicates high counts, and a - indicates low (background) counts.
STIMULATORS / TARGETS RESPONDERS / KILLERS

Mom

Dad

Duane

David

Mom Dad Duane David

+ + +

5. Which interpretation best explains these data? A. Both parents are heterozygous and share no HLA alleles. B. Both parents are homozygous and have identical HLA genotypes. C. Mom is homozygous and dad is heterozygous; but one of dads chromosomes has the same set of HLA alleles as moms. D. Dad is homozygous and mom is heterozygous; but one of moms chromosomes has the same set of HLA alleles as dads. E. The negative controls show this is a spurious result, and it could not be explained by any of the above choices. 6. Which statement about the two sons is true? A. Neither one could donate a graft to dad without rapid immunologic rejection. B. Either one could receive a graft from dad without rapid immunologic rejection. C. They are both heterozygous and are identical to one another at HLA. D. They are both homozygous and are identical to one another at HLA. E. They differ from each other at HLA, because humans are outbred. 7. Which of the sons have CD4+ CD8- T cells that could recognize nominal antigen processed and presented by moms APCs? A. Neither of them. B. Both of them. C. Only Duane. D. Only David. 8. Which of the sons have CD4+ CD8- T cells that could recognize nominal antigen processed and presented by dads APCs? A. Neither of them. B. Both of them. C. Only Duane. D. Only David.

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SHORT ANSWER QUESTION: Describe the molecular mechanisms operative in generating antibody diversity among primary B cells. Which of these operate in generating diversity within the T cell antigen receptor? Do all somatic mutations observed in the variable region during and immune response lead to higher affinity antibody of the same specificity? If not, speculate intelligently on why these are the antibodies that are observed to persist.

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