1. Innate Immunity (part II) and Antigen Recognition by Adaptive Immunity

2. Innate Immunity against viruses
Anti-viral immunity has 2 roles
Blocking infection (antibodies, complement, etc.)
Blocking viral replication (interferon, killing infected cells, anti-nucleic acid mechanisms)
Many viruses evolve extremely rapidly, great challenge for innate immunity
This problem is solved in part by a) targeting molecules that viruses have a hard time changing (dsRNA especially), and b) having multiple mechanisms, making it harder for a virus to evade all of them
Viruses are amazingly good at evasion of immune defenses, but often the most lethal viruses are those that are not well adapted to their host (their goal is to grow and be transmitted to others, not to kill)

3. INTERFERON: cytokine critical for defense against virus infections
Virus-infected cell produces interferon to act on neighboring cells
Uninfected cells respond to become refractory to viral growth (“anti-viral state”)
infected cell
Interferon-a
type 1 interferon= interferon a/b
type 2 interferon= interferon 

4. Induction of interferon by dsRNA in cytoplasm and RLRs
Mda5
IRF: interferon regulatory-factor
(family of transcription regulators)

5. Anti-viral effects of interferon a/b
Many other responses, less well understood
Note: PKR and OligoA
synthetase induced in
anti-viral state, but only
have enzymatic activity
following virus infection

6. Natural Killer (NK) Cells: innate lymphocytes that protect against intracellular infections
Abbas and Lichtman Fig. 2-10

7. NK cells are regulated by the balance between activating and inhibitory receptors
NK cell has activating receptors and inhibitory receptors: killing believed to occur if activating receptors dominate
(relative numbers of ligands on target cell)
stress-induced
proteins (red)
healthy cell
stressed cell

8. Summary of Innate Immunity to Viruses
Interferon (type 1) is absolutely critical for anti-virus immune defense; it is produced in two ways
by infected cells via RLRs or similar sensors of DNA for DNA viruses
by immune cells via nucleic acid-recognizing TLRs
Interferon induces anti-viral state, which is fully engaged by recognition of dsRNA in cytoplasm and inhibits virus replication (also promotes adaptive immunity)
Killing of infected cells is also performed by natural killer cells recognizing stress-induced molecules or loss of MHC class I molecule expression and by cytotoxic T cells which recognize virus antigens expressed by infected cells (+MHC I)

9. Pathogens and Innate Immunity
Particular pathogens often have evolved ways to evade at least some elements of innate immunity
Highly successful pathogens may also have mechanisms for evading adaptive immunity
On the pathogen side, molecules the pathogen uses to evade immunity are among the “virulence factors” of that pathogen

10. Part II: antigen recognition
Antigen recognition molecules: structure and function
B cell antigen receptors and T cell antigen receptors: Role in clonal selection
Classes of antibodies and their structures
Monoclonal antibodies and their uses in medicine (lab tests, therapies)

11. 2 Types of Antigen Recognition
1) Antibodies
Defense against extracellular microbes and viruses by soluble binding molecules linked to effector functions
B cells also make a membrane-bound form of immunoglobulin that serves as their antigen receptor
2) T cell antigen receptor
Peptide recognition mechanism for detecting antigens inside cells that are displayed on cell surface as peptides bound to MHC molecules

12. The B cell antigen receptor is membrane Ig + signaling chains
Antigen binding to more than one membrane Ig molecule triggers signaling reactions; tells B cell that antigen is present, induces activation (“clonal selection”)
Iga/Igb cytoplasmic domains have signaling function (ITAM sequence)
Iga/Igb
Signal transduction

13. The TCR also has ligand-binding chains + signaling chains
Figure 4-1 Abbas & Lichtman

14. Antibodies = immunoglobulins
Secreted by B lymphocytes (“plasma cells”)
Great diversity and specificity: >109 different antibodies; can distinguish between very similar molecules
Block infectivity of viruses, action of toxins (“neutralization”)
Tag particles for clearance/destruction (activate complement, “opsonize”for phagocytosis)
Protect against re-infection (vaccines)

15. Antibody Structure 2 identical heavy chains and
2 identical light chains
Both made of repeating structural unit: The Ig domain
Light chain
k or l
Ig domain: 110 amino acids; globular domain used in many proteins
Heavy chains

16. Antibody Structure
Domains at ends of heavy chain and light chain are highly variable and responsible for binding antigen

18. Variability in antibodies is clustered in the loops in the variable domains of the heavy and light chains (green); these regions are responsible for binding to antigen
(“hypervariable regions”; “complementarity-determining regions”)
Fab, variable domains in color

20. Similarities between Ig and TCR
Both are composed of Ig domains
Both have two variable subunits (H + L for Ig; TCR a and TCR b; there are also “gd T cells”)
Both have great diversity and exquisite specificity
Both recognize antigen via hypervariable loops at the ends of the variable domains
Both couple antigen recognition to lymphocyte activation (“clonal selection”) via signaling chains with very similar signaling mechanisms (ITAM sequences)
(Only Ig is secreted and only Ig has class switching to change the constant domains)

21. 5 Different Antibody Classes
Heavy chains are named by the greek letter corresponding to the type of antibody (m, g, d, a, e)
All classes can have k or l light chains

22. Antibody Classes II: Distinctive features
IgM
First antibody produced in immune response: multimeric structure adds to “avidity” for antigen
Antigen receptor of naïve B cells
IgD
Used primarily as antigen receptor, little secreted
IgG
Major antibody of serum and extravascular tissues
Crosses placenta to provide protection in utero
IgA
Major antibody of secretions (mucus, saliva, milk, etc.)
IgE
Important for immune defense against helminths; allergy and asthma

23. Affinity and Avidity
Affinity: the strength of binding between a single binding site and a single ligand
Avidity: the strength of binding between a molecule and a complex ligand. If there are multiple binding sites then the avidity may be increased by increasing the number of binding sites or by increasing the affinity of those binding sites

24. Affinity and Avidity II
IgM is produced early in an immune response when the affinity for antigen often is low; as an immune response continues, antibody affinity is improved, this is combined by “class switching” to the use of smaller molecules (IgG, IgE and IgA). The increased affinity compensates for the decrease in number of binding sites in maintaining the overall avidity for antigen

25. Antibody Classes II: Distinctive features
IgM
First antibody produced in immune response: multimeric structure adds to “avidity” for antigen
Antigen receptor of naïve B cells
IgD
Used primarily as antigen receptor, little secreted
IgG
Major antibody of serum and lymph
Crosses placenta to provide protection in utero
IgA
Major antibody of secretions (mucus, saliva, milk, etc.)
IgE
Important for immune defense against helminths; allergy and asthma

26. Antibodies and medicine
Vaccination works by inducing production of protective antibodies
Antibodies from immunized or pooled donors (“IVIG”) can provide “passive immunity” (used for tetanus, snake bites, etc.; to treat immunodeficiencies)
Antibodies are often used to diagnose infectious diseases (e.g., presence of antibodies in patient’s blood), determine bloodtype, diagnose type of cancer, etc.
Increasingly, antibodies are used to treat diseases (cancer, autoimmune disease, etc.): advantage of monoclonal antibodies

27. Monoclonal Antibodies
Normal antibodies are “polyclonal”, as they are mixtures of antibodies made by several different clones of B cells
Monoclonal antibodies: Single antibody (all same H and L chains): more reliable, consistent; can be produced in unlimited quantities
Made by fusion of B cells to a transformed cell line of the plasma cell type and selection for “hybridomas” that produce antibody with the desired properties

28. Use of antibodies in lab tests: immunoprecipitate-based tests
(Polyclonal antibodies)

29. Use of antibodies in lab tests: ELISA
Enyzyme-linked immunosorbant assay (ELISA): measure levels of antibody or antigen, depending on assay design (“sandwich ELISA is shown)

30. Use of antibodies in lab tests: flow cytometry
Flow cytometry: measure the amount of a protein on the surface (or inside) individual cells; measure the numbers of particular types of cells in blood (etc.)

31. Monoclonal antibodies used in medicine
Standardized, unlimited reagents for diagnosis or therapy
(human antibodies or “humanized” antibodies can be made)
The list of monoclonal antibodies FDA-approved for therapy is increasing by several per year
Names: fully human: -mumab
humanized: -zumab
chimeric: ximab
murine: omab

32. Monoclonal antibodies as therapeutics
A potential problem, as with other protein therapeutics, is immunogenicity, a fraction of patients make an antibody against the therapeutic. This can make the therapeutic ineffective in those individuals.
In general, the more “human” the monoclonal antibody, the less immunogenicity, but this is not a perfect correlation (see syllabus for further discussion of this issue).

33. Anti-TNF therapeutics
TNFR2
Adalimumab (Humira)
etc.
Infliximab, etc.: anti-TNF monoclonal antibodies (+ 3 others)
Etanercept: fusion between TNF receptor extracellular domain and Fc part of IgG1, which greatly extends half-life in serum
These agents are used to treat Rheumatoid arthritis, Crohn’s disease, etc.