Antigens

ASSIGNED READING CHAPTER: 4 pp 87-106
OUTLINE/SUMMARY

1. Immunogenicity vs antigenicity
2. Factors that influences immunogenicity
   • immunogen contributions
   • biological system contributions
3. Epitopes
   • B cell epitopes
   • T cell epitopes
4. Haptens
5. Mitogens

PERFORMANCE OBJECTIVES

DEFINE THE FOLLOWING KEY TERMS

ON COMPLETION OF THIS SECTION THE STUDENT SHOULD BE ABLE TO:

1. Differentiate between and antigen's inductive and reactive abilities.
2. Explain the factors that bestow immunogenicity on molecules
3. Describe the discrete and distinctive sites on and antigen that are recognized by a B cell
4. Immunoglobulin
5. Describe the discrete and distinctive sites on and antigen that are recognized by a T cell receptor
6. Describe the discrete and distinctive sites on and antigen that are recognized by the Major
7. Histocompatability Complex
8. Discuss some important characteristics of protein antigenic determinants

SHORT ANSWER QUESTIONS

1. Differentiate between immunogenicity and antigenicity.
2. All immunogens are antigens but all antigens are not immunogens. Explain with the use of specific examples.
3. An entire microbe or molecule does not induce and immune response. Explain.
4. Irrespective of the number of epitopes that a molecule has, the immune response is biased and sees what it wants to see. Explain.
5. Haptens are usually coupled to a carrier molecule before they are immunogenic. How can you demonstrate that the selected immunoglobulins are specific for the hapten and not for the carrier? As a corollory, would you expect ALL of the antibodies to be specific for the hapten? explain.
6. Why is the definition of an antigen considered to be an operational definition.
7. Differentiate between epitopes and agreotopes.
8. Differentiate between epitopes recognized by B cell receptor and epitopes recognized by T cell receptors.

Immunoglobulin Structure & Function

ASSIGNED READING CHAPTER 5: pp 107-142
OUTLINE/SUMMARY

1. Basic Structure
2. Immunoglobulin Sequencing Studies
3. Immunoglobulin Fine Structure
4. B Cell Receptor
5. Antigenic Determinants of Igs
6. Ig Classes
7. Immunoglobulin Superfamily
8. Monoclonal Antibodies (MABS)

PERFORMANCE OBJECTIVES

DEFINE THE FOLLOWING KEY TERMS:

ON COMPLETION OF THIS SECTION THE STUDENT SHOULD BE ABLE TO:

1. Distinguish between the overall structure and the fine structure of immunoglobulins
2. Describe the variable and constant regions of immunoglobulins light and heavy chains
3. Explain the structural organization of the variable regions of an immunoglobulins light and heavy chains
4. Differentiate between Hypervariable regions and complementarity determining regions
5. Contrast monoclonal antibody production vs polyclonal antibody production
6. Discuss the differences in the biological effector functions of the different classes of immunoglobulins
7. Diagram the procedure for monoclonal antibody production
8. Construct a table comparing the biological characteristics of the five classes of immunoglobulins
9. Construct a table comparing the biochemical and biophysical characteristics of the five classes of immunoglobulins

SHORT ANSWER QUESTIONS

1. The analysis of IgG molecules after hydrolysis by pepsin and papain led to similar yet different results. Explain.
2. What is the difference between and immunoglobulin and a myeloma protein?
3. Why were myeloma proteins and Bence Jones proteins critical to the early studies on antibody structure? What has largely replaced them?
4. Differentiate among, complementarity determining regions, hypervariable regions and framework regions.
5. Explain the statements:
   • "Antibodies can be antigens" and
   • "Antibodies can be used to characterize antibodies".
6. Discuss the terms immunoglobulin isotypes, immunoglobulin allotypes and immunoglobulin idiotypes and give examples of each.
7. Compare conventional antibody production with monocloanl antibody production and recombinant antibody production.
8. What are antibody domains?
9. Why can't light chains be used to classify antibodies?
10. The function of an immunoglobulin molecule is related to its structure. Explain.
11. Different cellular fusion products can potentially occur in a hybridoma fusion.
    • What are the potential products?
    • Why are they a problem?
    • How can you obtain the appropriate combination?
12. Distinguish between idiotypes and anti-idiotypes.

Antigen-Antibody Interactions

ASSIGNED READING CHAPTER 6: pp 144-164
OUTLINE/SUMMARY

1. Strength of Antigen-Antibody Interactions
   • affinity
   • avidity
2. Cross Reactivity
3. Precipitin Reactions
   • reactions in fluids
   • reaction in gels
   • radial immunodiffusion
   • Ouchterlony
   • immunoelectrophoresis
4. Agglutination Reactions
   • hemagglutination
   • bacterial agglutination
   • passive agglutination
   • agglutination inhibition
5. Radioimmunoassay (RIA)
6. Enzyme Linked Immunosorbent Assay (ELISA)
   • indirect ELISA
   • sandwich ELISA
   • Competitive ELISA
7. Western Blots
8. Immunofluorescence
9. Immunoelectron Microscopy

PERFORMANCE OBJECTIVES

DEFINE THE FOLLOWING KEY TERMS

ON COMPLETION OF THIS SECTION THE STUDENT SHOULD BE ABLE TO:

1. Explain a primary antigen-antibody interaction and include at least three important charactistics
2. Describe the forces that encourage primary antigen-Antibody interactions
3. Distinguish betweeen antibody affinity and avidiy.
4. Describe the strength of the primary antigen-Antibody interactions using equilibrium dialysis. Include the terms K and Ka
5. Compare and contrast RIA and ELISA
6. Describe direct and indirect fluorescent antibody methods.
7. Describe a secondary antigen-antibody interaction in terms of lattice formation and antigen:antibody ratios.
8. Explain zone phenomena.
9. Construct a table to compare the various procedures used to determine the presence of solbule antigen or antibody in a fluid and in a gel.
10. Assess the reasons for using the different gel preciptitin reactions
11. Distinguish between agglutination and preciptin reactions and give the advantages and disadvantages of each.

SHORT ANSWER QUESTIONS

1. Cross reactivity of antibodies creates problems for their application in serology. Explain.
2. Differentiate between a primary and a secondary antigen-antibody reaction. What are three important characteristics that distinguish the two reactions?
3. What kinds of noncovalent interactions are important in antigen-antibody interactions? What aspect of these interactions is most important and why?
4. How is equilibrium dialysis used to measure PRIMARY antigen-antibody reactions?
5. Differentiate between avidity and affinity.
6. Discuss the term lattice formation.
7. What are the pros and cons of RIA?
8. Describe two types of immunofluorescence tests.
9. What is the advantages of the indirect procedure over the direct procedure?
10. What are some commonly used fluors?
11. What colour does each fluor emit?
12. What makes precipitin reactions visible?
13. What two factors are important in the development of precipitin reactions?
14. Three patterns can be observed in the Ouchterlony test.
15. DRAW and LABEL diagrams to illustrate these patterns.
16. What does each pattern show?
17. What is the major advantage of immunoelectrophoresis over immunodiffusion?
18. What are the disadvantages?
19. How does agglutination differ from precipitation?
20. Why are agglutinatin tests more sensitive that precipitin tests?
21. Differentiate between direct and indirect agglutination reactions?
22. What is a major advantage of indirect agglutination reaction over direct reactions?

Immunoglobulin Genes

ASSIGNED READING CHAPTER 7: pp 165-194

OUTLINE/SUMMARY

1. Genetic models
   • germ line vs somatic mutation model
   • Dryer Bennett: Two gene model
   • Verification
2. Multigene Organization of Immunoglobulin Genes
   • lambda chain multigenes
   • kappa chain multigenes
   • heavy chain multigenes
3. Variable-Region Gene Rearrangements
   • V-J rearrangements of light chains
   • V-D-J rearrangements of heavy chains
   • mechanisms
   • recombination signal sequences
   • enzymatic joining of gene segements
   • RAG-1 RAG-2
   • Defects
   • Productive vs non productive gene rearrangements
   • Allelic exclusion
4. Generation of Antibody Diversity
   • multple germ line gene segments
   • combinatorial V-J & V-D-J joining
   • junctional flexibility
   • P-nucleotide addition
   • N-nucleotide addition
   • Somatic hypermutation
   • Assocation of light & heavy chains
5. Class Switching in C Region Genes
   • Expression of Ig Genes
     • Differential processing of RNA transcipts of heavy chains
       • Membrane bound vs secreted Ig
       • Coexpression of IGM & IgD
       • Synthesis, assembly and secretion
   • Regulation of Ig-Gene Transcription
     • DNA rearrangement
     • Inhibition by T Cells

PERFORMANCE OBJECTIVES

DEFINE THE FOLLOWING KEY TERMS:

ON COMPLETION OF THIS SECTION THE STUDENT SHOULD BE ABLE TO:

1. Describe how the number and organization of immunoglobulin gene segements or exons contribute to the generation of antibody diversity
2. Draw the organization of Kappa light chain genes.
3. Draw the organization of Lambda light chain genes.
4. Draw the organization of heavy chain genes.
5. Describe the V-J joining for light chain genes in terms of the 7-9 and the 12-23 rules
6. Describe the V-D-J joining of heavy chain genes in terms of the 7-9 and the 12-23 rules.
7. Explain how allelic exclusion leads to cells that produce immunoglobulins with a single antigen binding site.
8. Describe how transcription controls immunoglobulin gene expression
9. Describe at least four mechanisms that contribute to antibody diversity
10. Describe the coexpression of IgM and IdD in terms of RNA processsing
11. Describe the mechanisms for class switching
12. Correlate B cell differentiation with immunoglobulin gene rearrangements

SHORT ANSWER QUESTIONS

1. Why is it important to have antibody diversity?
2. Differentiate between the germline theory and the somatic theory of generation of antibody diversity.
3. How are BOTH germline and somatic mechanisms sources of antibdoy diversity?
4. Why did the amino acid sequencing of antibody light chains lead Dryer and Bennet to abandon the one gene- one polypeptide theory and propose that two genes code for a single polypeptide?
5. Describe Tonagawa's approach to answering Dreyer and Bennett's hypothesis that two genes code for a single polypeptide.
6. In addition to showing that DNA seqments come together to form a functional antidoby gene, Tonagawa's experiments showed two other things. What were they?
7. The phrases "race car", "Madam I am Adam" and "sex at noon taxes" represent pallindromes in the english language. What are pallindromes in the genetic code and what is their importance in V region construction?
8. What are the two DNA recombinational events required to assemble a functional heavy chain V region gene?
9. The two DNA recombinational events required to assemble a functional heavy chain V region gene, are governed by the 7-9 and the 12-23 rules. Explain.
10. Discuss four mechanisms that contribute to antibody diversity.
11. Describe the differential splicing of RNA that progresses from membrane bound IgM to secreted IgM.
12. Explain how a particular B cell can secrete different antibody isotypes, yet remain specific for the same epitope.
13. What are the two stages of class switching?
14. Dicuss allelic exclusion; what it is and how it is regulated.
15. List in correct chronological order the events leading to the synthesis of a Kappa light chain and its incorporation into an antibody molecule.
16. List in correct chronological order the events leading to the synthesis of a Gamma chain and its incorporation into an antibody molecule.

B Cell Maturation

ASSIGNED READING Chap 8 pp 195-222

OUTLINE/SUMMARY

1. B Cell Maturation
   • Bone marrow
   • Ig Gene rearrangements
   • Pre B cell receptor
   • Selection of Immature Self Reactive B Cells
   • negative selection
   • rescue editing of light chain genes
2. B Cell Activation & Proliferation
   • T dependent vs T independent antigens
   • Origins of activating signals
   • Transduction of Activating signals
   • B cell coreceptor complex
   • T helper cells
     • formation of T-B conjugate
     • mediation by CD40
     • induction of progression signals
     • Negative selection
3. In Vivo induction of Humoral Response
4. B Cell Differentation
   • Affinity maturation
     • somatic hypermutation
     • antigen selection
     • Class switching
     • Plasma cells
     • Memory cells
5. Regulation of B Cell Development

PERFORMANCE OBJECTIVES

DEFINE THE FOLLOWING KEY TERMS:

ON COMPLETION OF THIS SECTION THE STUDENT SHOULD BE ABLE TO:

1. Describe the sequential development of the humoral immune response.
2. Draw a contemporary model for T cell B cell macrophage interaction
3. Describe antigen processing and presentation by B cells
4. Outline the sequence of helper (T/(TH) cell involvment in antibody production
5. Describe the interplay between TH1 and Th2 cells in the development of immune responses

QUESTIONS

Major Histocompatability Complex

ASSIGNED READING CHAPTER 9:pp 223-248

OUTLINE/SUMMARY

1. General Organization of MHC
   • Location & function of MHC regions
   • MHC haplotypes
   • Congenic mice
2. MHC Molecules & Genes
   • Structure of MHC-I
   • Structure of MHC-II
   • Gene Organization
   • Peptide Binding
   • MHC-I
   • MHC-II
   • Polymorphism
   • ClassII molecules
3. Genomic maps of MHC
   • MHC-I
   • MHC-II
   • MHC-III
4. Cellular Distribution of MHC
5. Regulation of MHC Expression
6. MHC and Immune Responsiveness
   • Determinant selection model
   • Hole in the repertoire model
   • MHC and Disease

OBJECTIVES

DEFINE THE FOLLOWING KEY TERMS

ON COMPLETION OF THIS SECTION THE STUDENT SHOULD BE ABLE TO:

1. Explain the connection between a trait and a gene using the specific example that MHC genes encode for molecules that allow specific immune responses.
2. Describe the use of inbred mice for the elucidation of the genetics of the imune response
3. Describe how congenic strains of mice are developed.
4. Describe and draw MHC-I molecules
5. Describe the function of MHC-I molecules
6. Describe and Draw MHC-II molecules
7. Compare the detailed differences in the structure of the aggreotope binding sites of MHC-I and MHC-II
8. Describe the function of class III genes and in which cells they are expressed.
9. Describe MHC restriction
10. Discuss the differences between MHC restriction of CD4+ T cells and MHC restriction of CD8+ T cells.
11. Draw maps of either the MHC gene complex in mice or HLA gene complex in humans
12. Describe how the collection of MHC genes of an individual dictates either the ability or inability of the animal to respond to a particular antigen
13. Describe an experiment that shows MHC restriction: between T and B cells, between T cells and macrophages between cytotoxic T cells and virally infected cells.
14. Discuss the biological relevance of MHC molecules
15. MHC molecules exhibit a range of structural diversity at the species level that is roughly equivalent to the range of antibody diversity at the level of the individual animal. DISCUSS

SHORT ANSWER QUESTIONS

1. Null cells are not MHC restricted. Explain.
2. Even though the immune system rejects transplanted kidneys and hearts its
3. function is not to protect us against grafts.
4. Why do we need histocompatability antigens?
5. If we do not need protection against attack from foreign organs and tissues, why are MHC-I molecules so polymorphic?
6. What is the connection between immunity and MHC genes.?
7. Why are inbread/congenic mice important to immunlogical studies?
8. Draw and label a diagram of a class I MHC molecule as it is found in the membrane.
9. Draw and label a diagram of a class II MHC molecule as it is found in the membrane.
10. How is the polymorphism or diversity of MHC, different from the generation of diversity in antibodies?
11. How do MHC-II molecules allow immune cells to communicate with each other? Why is this communication important?
12. What is the relationship between MHC-II molecules and Ir genes?
13. Class III MHC molecules are not cell membrane proteins.
14. What are they and what do they do?
15. Describe an experiment to show MHC restricted cytotoxicity
16. T-cells do not recognize free antigen, as antibody receptors do. Speculate why?
17. Briefly discuss MHC restriction.
18. Describe and experiment that describes MHC restriction.
19. Why do T cells have such an elaborate way of reacting with antigen?
20. Explain the following statement:
21. "There is a direct relationship between an animal's MHC haplotype and its ability or inability to respond to a specific antigen."
22. What is the difference between and F1 hybrid animal and a bone marrow chimera animal?
23. What is the importance of a bone marrow chimera animal in immunological studies?
24. Explain how the control of MHC restriction is the genetic basis for the functional specificity of T cells.
25. Describe T cell differentiation in the thymus using CD4 and CD8 markers.
26. T cell receptor diversity results from gene rearrangements, but T cells are also MHC restricted. MHC restriction is not preporgrammed into T cells. Describe two possible selective processes that program T cells to be MHC restricted. Where do these processes occur?
27. T cells can react only with protein fragments. What is this process called? How does it occur? Which pathway leads to antigen interaction with MHC-I molecules? Which pathway leads to interaction with MHC-II molecules?

Antigen Processing

ASSIGNED READING Chapter 10: pp 249-262

OUTLINE/SUMMARY

1. Self MHC Restriction of T Cells
2. Role of Antigen Presenting Cells
3. Two Presentation Pathways
4. Cytosolic Pathway: Endogenous Antigens
   • proteasomes
   • peptide transport
   • assembly of peptides in MHC-I
5. Endocytic Pathway: Exogenous Antigens
   • endocytic vesicles
   • peptide transport
   • Assembly of peptides in MHC-II
6. Clinical Applications

OBJECTIVES

DEFINE THE FOLLOWING KEY TERMS:

ON COMPLETION OF THIS SECTION THE STUDENT SHOULD BE ABLE TO:

1. Describe self MHC restriction.
2. Compare the cytosolic pathway for antigen presentation with the endocytic pathway for antigen presentation
3. Compare profession with non professional antigen presenting cells.
4. Draw a flow diagram comparing cytosolic and endocytic pathways for processing antigens [Fig 10-4]
5. Describe peptide generation by proteasomes.
6. Describe transport of antigenic peptides from the cytosol to the Rough Endoplasmic reticulum
7. Draw a concept map for the separate antigen presenting pathways for endogenous and exogenous antigens.
8. Describe the assembly of MHC-II molecules within the Rough endoplasmic reticulum.
9. Describe the development of a viral vaccine that uses the cytosolic pathway.
10. Describe the development of a vaccine that used the endocytic pathway.

SHORT ANSWER QUESTIONS

1. Name thee types of professional APCs.
2. For each type indicate whether it expresses MHC-II molecules and a co-stimulatory signal constitutively or must be activated before doing so.
3. Describe the role of ubiquitin in the processing of endogenous antigens.
4. Describe the role of calnexin in the assembly of MHC-I.
5. T cells can react only with protein fragments.
   • What is this process called?
   • How does it occur?
   • Which pathway leads to antigen interaction with MHC-I molecules?
   • Which pathway leads to interaction with MHC-II molecules?

T Cell Receptor

ASSIGNED READING CHAPTER 11: pp 264-283

OUTLINE/SUMMARY

1. Early Studies
   • self MHC restriction
   • isolation of t cell receptors
2. Structure of T cell Receptors
3. TCR Genes
   • Cloning
   • TCR multi gene families
   • Variable region rearrangements
   • TCR DNA rearrangements
   • allelic exclusion
   • Structure of rearranged TCR genes
   • Generation of TCR diversity
4. T Cell Receptor Complex
   • CD4 & CD8 coreceptors
   • Accessory membrane molecules
5. TCR-Peptide-MHC Complex
   • Associative recognition hypothesis
   • Formation of ternary complex
   • Affinity of TCR
   • Peptide topology
6. Alloreactivity of T Cells

OBJECTIVES

DEFINE THE FOLLOWING TERMS

ON COMPLETION OF THIS SECTION THE STUDENT SHOULD BE ABLE TO:

1. Draw the structure of the T Cell Receptor (TCR)
2. Compare and constrast the structure of TCR with B cell receptor
3. Draw a diagram of the T cell receptor gene organization
4. Describe the stragetgies for diversification of T Cell Receptors
5. Describe how a T cell Receptor recognizes an antigen
6. Describe the TCR-Antigen-MHC complex

QUESTIONS

1. Hedrick and Davis used the technique of subtractive hybridization to isolate cDNA clones encoding for the T cell receptor.
2. Briefly explain the method of subtractive hybridization.
3. State why this method was so important in the characterization of the TCR.
4. What does it mean to say that TCRs belong to the Ig supergene family? Briefly describe the heterodimeric structure of the TCR.
5. TCR genes use the same strategies as antibody genes to develop receptor diversity; however, TCR genes do not exhibit somatic hypermutation. Why?
6. The TCR is closely assoicated with another surface protein compolex. What is its name and function?
7. Describe the three way complex that occurs during antigenic recognition by T cells.
8. T cells can react only with protein fragments.
   • What is this process called?
   • How does it occur?
   • Which pathway leads to antigen interaction with MHC-I molecules?
   • Which pathway leads to interaction with MHC-II molecules?
9. What are accessory molecules?
10. Give some examples.
11. briefly discuss how they may be involved in antigen recognition by T cells.

T cell Maturation

ASSIGNED READING CHAPTER 12: pp 285-310

OUTLINE/SUMMARY

1. T Cell Maturation
   • Pre T cell receptor
   • T cell development pathways
   • Thymic Selection of T cells
   • positive selection
   • negative selection
2. T helper Cell Activation
   • TCR coupled signaling pathways
   • Co-stimulatory signal
   • Antagonists & Agonists
   • Clonal Expansion
   • Clonal Anergy
   • Superantigens
3. T Cell Differentiation
   • Effector & memory cells
   • Co-stimulatory differences
4. Peripherial gamma delta T cells
   • Distribution of cells
   • Ligands recognized
   • Functions

PERFORMANCE OBJECTIVES

DEFINE THE FOLLOWING KEY TERMS:

ON COMPLETION OF THIS SECTION THE STUDENT SHOULD BE ABLE TO:

1. Outline antigen processing and presentation to TCR
2. Describe the molecular pathways for T cell activation
3. Describe positive and negative selection
4. Discuss the role of accessory molecules in T cell antigen recognition

SHORT ANSWER QUESTIONS

1. T cell proliferation and differentiation lead to important immunological functions. What are they?
2. Briefly discuss the molecular events, collected called T-Cell activation, that lead to biologic function.
3. What is the importance of diacylglycerol and IP3 in T-cell activation?
4. What transcription factors are involved in T cell activation?