1. Viral Immunity
Viruses evolve extremely rapidly, great challenge for innate immunity
Anti-viral immunity has 2 roles
Blocking infection (antibodies, complement, etc.)
Blocking viral replication (interferon, killing infected cells)
Viruses have evolved many mechanisms of evading immunity

2. Anti-retroviral defense by a cytidine deaminase APOBEC3G
HIV Vif binds to APOBEC3G and blocks it
(details of virus assembly not accurate)

3. Virus-infected cell produces interferon to act on neighboring cells
Interferon-a
Infected cell makes interferon, uninfected cells respond to interferon and become refractory to viral growth
Virus-infected
cell

4. Production of interferon by infected cells
RIG-I, MDA-5: have
RNA helicase domain
and CARD domain (“RLRs”)
Required adaptor “MAVS”

5. Intracellular sensing mechanism for DNA virus infection
O’Neill, L.A.J.
Science 339: 763-4, 2013

6. Response of cells to interferons

7. Anti-viral effects of interferon a/b
© New Science Press Ltd. 2004

8. Viral evasion of interferon: PKR
© New Science Press Ltd. 2004

9. Plasmacytoid dendritic cells
Many cell types produce small amounts of type 1 interferons upon infection
There is a dendritic cell subtype (“plasmacytoid dendritic cell”; “natural interferon-producing cell”) that produces x more interferon upon contact with viruses, does not need a productive infection.
Also produces a large amount of TNF
Recognition mechanism: probably TLR7, TLR9
Function? anti-viral innate immunity; stimulation of adaptive immunity?

10. 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

11. Antibodies Secreted by B lymphocytes
Great diversity and specificity: >109 different antibodies; can distinguish between very similar molecules
Tag particles for clearance/destruction
Protect against re-infection (vaccines)

12. Antibody Structure
Ig domain: 110 amino acids; globular domain used in many proteins;
Variable domains,
Constant domains, Hinge
Fab: fragment antigen binding
Fc: fragment crystallizable (effector functions)

14. Antibody Structure supports Function
Variability in antibodies is clustered in the loops in the Variable domains of the heavy and light chains (green);
Green areas are called: hypervariable regions or complementarity determining-regions

15. The Immunoglobulin Superfamily (a few examples)
Ig and TCR are very similar structurally

16. Antibody Classes: Structure

17. Affinity and Avidity
Affinity: the strength of binding between a single binding site and a single ligand
[A][B]
[AB]
Avidity: the strength of binding between a molecule and a complex ligand, e.g. 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 KD =

18. 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

19. Antibodies can be directly protective or can promote immune protective mechanisms via other cells or molecules
neutralization
activation of complement

20. (?)

21. Fc receptors contribute to effector functions of antibodies
human
There are a number of activating FcRs, which utilize ITAM signaling (the two hexagon motif represents the ITAM)
There is also an inhibitory FcR (IIB), which has an ITIM that inhibits ITAM signaling
There are transport FcRs for IgA and IgG.
Effector functions: phagocytosis, degranulation, antibody-dependent cytotoxicity
a chains are unique in each case; g chains are the same.

22. Monoclonal Antibodies
Single antibody (all same H and L chains)
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
Standardized, unlimited reagent for diagnosis or therapy (human antibodies or “humanized” antibodies can be made)

23. Polyclonal vs. Monoclonal Antibodies
Immunize Individual
B lymphocytes
3 days
7+ days
Immortalize
Serum
Purify
antibodies
“hybridoma”
cDNA cloning
Polyclonal antibodies
Monoclonal Antibodies

24. Generation of Monoclonal Antibodies

25. Monoclonal antibodies used in medicine
Standardized, unlimited reagents for diagnosis or therapy
(human antibodies or “humanized” antibodies can be made)

26. Generation of antibody diversity
How do we make >109 different antibodies?
Genes for antibodies are present in pieces that can be combined in many different combinations in different lymphocytes
V region of Ig L and H chains are constructed from 2 and 3 different pieces each having multiple copies

27. V(D)J recombination

28. Generation of Antibody Diversity
k light chains: 40 Vk x 5 Jk = 200
l light chains: 30 Vl x 4 Jl = 120
H chains: 40 VH x 27 DH x 6JH = 6,480
320 L chains x 6,480 H chains = 2.1 x 106 (“Combinatorial diversity”)
Also there is “Junctional diversity” (addition or deletion of nucleotides at recombination sites, especially of H chain), estimated to add substantially to overall diversity

29. Discovery of Rag1, 2 genes
“Recombination Activating Gene”

30. Mechanism of V(D)J recombination
CACCGTG
Recombination signals (12/23 rule)
Rag-1/Rag-2/Artemis
DNA repair proteins
(non-homologous end joining proteins)
(defects: Severe Combined
Immunodeficiency (SCID))

31. Creation of Junctional Diversity by P-regions and TdT
(There can also be trimming back by nucleases before addition of N regions)
Key point: reading frame must be preserved to get functional gene

32. Defects in Lymphocyte development leading to severe combined immunodeficiency (SCID)
Note: SCID can also result from defects that interfere with lymphocyte development (adenosine deaminase deficiency, purine nucleotide phosphorylase deficiency, MHC defects, etc.)

33. Lymphoid malignancies resulting from errors in V(D)J recombinationCH 4
VDJ Recombination reactions contributes to translocation leading to over-expression of a cellular growth or survival promoting gene CH 4

34. Ig Heavy chain class (isotype) switching

35. Affinity maturation and antibody responses

36. Ig mutations are localized near transcription start site
from Longacre and Storb Cell 102: 541, 2000.

37. Comparison of VDJ recombination, class switch recombination and somatic hypermutation
ssDNA
nicks?

38. Comparison of VDJ recombination, class switch recombination and somatic hypermutation
ssDNA
nicks?

39. Activation-induced cytidine deaminase (AID)
Discovered as an induced gene in a cell line with inducible class-switch recombination (subtractive hybridization)
Transfection into B cell lines induces class switch recombination
AID KO mice have strong defect in class switch recombination AND in somatic hypermutation
Hyper-IgM syndrome type 2 (autosomal) is due to mutation in AID; very similar phenotype to mice (no IgG, IgA, IgE; very much reduced somatic mutation)

40. AID: How does it work?
AID is highly related to APOBEC-1, a cytidine deaminase that edits mRNA for Apolipoprotein B (via a targeting subunit)
indirect action or direct action in class switch and hypermutation?
AID could edit mRNAs for factors that act in class switch and factors that act in class switch OR
it could act directly in both processes

41. AID as a mutator of DNA
AID is mutagenic in bacteria and mutations are increased by deficiency in Uracil-DNA glycosylase (enzyme that removes U from DNA and triggers DNA repair)
Class switch is inhibited and hypermutation perturbed in UNG-deficient mice
These results favor the hypothesis that AID directly acts on C residues in DNA to promote class switch and hypermutation

42. Model for direct actions of AID in somatic mutation and class switch
In hypermutation:
U in DNA could lead to direct mutations and secondary mutations via mismatch repair and/or error-prone DNA polymerases
In class switch recombination: U in DNA could lead to nick formation by repair enzymes:
nicks on both strands-->ds breaks-->recombination