3. Antigen Processing and Presentation
Dr. M.S.A. Muthukumar Nadar,
Assistant Professor,
Dept of Biotechnology,
Karunya University,
Coimbatore

4. What does the immune system do?
The immune system protects us from infections:
~ 208 viruses
~ 538 bacteria
~ 317 Fungi
~ 287 Worms
~ 57 Parasitic Protozoa
Promote normal functioning of the body such as Tissue cleanup, wound healing
Removes abnormal cells –including cancer cells
But the immune system can also harm the body when not doing the right thing (Allergy, Auto immunity, Transplanted organ rejection, Immuno-deficiency

5. Distinguishing SELF AND NON-SELF

6. IMMUNE SYSTEM DESIGN IDENTIFICATION Non-infectious Self and
infectious Non-self
RECRUITMENT
Mobilize Effectors
ELIMINATION
Clears Pathogen
PREVENT RECCURENCE
Prevention of Future infection-(Memory)
DEACTIVATION
Stop fighting when the war is over-Prevention of unwanted effects on host (Reducing Collateral damage)

7. Components of the immune system
Innate immune system
Adaptive immune system
Response is non-specific
Pathogen and antigen specific response
Exposure leads to immediate maximal response
Lag time between exposure and maximal response
Cell-mediated and humoral components
No immunological memory
Exposure leads to immunological memory
Found in nearly all forms of life
Found only in jawed vertebrates

8. Jawless vertebrates (Agnatha) Lamprey

10. Hagfish: Notice the mouth- No Jaws

11. Surface Barriers
Skin
Skin is a barrier that protects and seals the organisms from infection that could be caused by mechanical injury, chemical injury, and biological injury.

12. Composition of Blood
PELLET
19-12

13. Pathogen-associated molecular patterns- PAMPs
PAMPs are molecules associated with groups of pathogens, that are recognized by cells of the innate immune system.
These molecules can be referred to as small molecular motifs conserved within a class of microbes.
They activate innate immune responses, protecting the host from infection, by identifying some conserved non-self molecules.

14. PAMPs & Pattern Recognition Receptors
Bacterial Lipopolysaccharide (LPS)
TLR 4
Flagellin
TLR 5
Peptidoglycan
Lipoteichoic acid (Gram positive bacteria)
Viral dsRNA
TLR 3
Unmethylated CpG motifs
TLR 9

15. Leukocytes Have nucleus, mitochondria, & amoeboid ability
Can squeeze through capillary walls (diapedesis or extravasation)
Granular leukocytes help detoxify foreign substances & release heparin
Include eosinophils, basophils, & neutrophils
13-10

16. Leukocytes Agranular leukocytes are phagocytic & produce antibodies
Include lymphocytes & monocytes

18. Types of T Lymphocytes
Helper T cells (CD4)-Activates B-cells (B-cell proliferation and antibody production)
Cytotoxic T cells (CD8)– attack and destroy invading agent or antigen
Suppressor T Cells (T-reg)– Inhibit or terminate activities of killer cells, plasma cells or T helper cells when their activities are no more needed

19. T Cell Recognition of Antigen
T-cells do not recognize PAMPs or whole pathogens.
They recognize antigen peptide fragments bound to specialized cell surface molecules on antigen-presenting cells (APC).
Peptides are displayed to T cells as peptide:MHC complexes
T cell antigen receptors recognize peptide:MHC complexes
Each MHC molecule can bind numerous different peptides

20. MHC restriction
The T cell receptor will recognise a peptide only when it is bound to a particular MHC molecule.
1973: Peter Doherty and Rolf Zinkernagel (Nobel Prize in Medicine, 1996)
MHC: Major Histocompatibility Complex
encoded by the most polymorphic gene cluster on the
human genome (many alleles)

21. Before being presented, antigens have to be processed such that they fit in the MHC molecule in a suitable way to be recognized by T-cells

22. Antigen presentation:
Antigen processing:
Proteolytic cleavage of proteins into small fragments (antigen peptides) that can bind to MHC molecules on antigen presenting cells.
Antigen presentation:
Presentation of antigen peptides to T cell receptor on T cells

23. Ag is processed through 2 separate pathways:
Ag is processed through 2 separate pathways: *MHC I interacts w/ peptides from cytosolic degradation *MHC II interacts w/ peptides from endocytic degradation

24. Processing Endogenous Antigens: the Cytosolic pathway
Cellular proteins are constantly regulated; most have a brief half-life and are “turned over”… the same holds true for endogenous Antigens!
Processing of endogenous Ag involves 3 processes:
Peptide generation from proteolysis
Transport to ER
Loading peptide on to MHC I

27. MHC-I
See figure 8-3,
Kuby, 6th Edition,
p. 194, left

28. Class I MHC Protein Binding Cleft

30. 10 A

31. ~ 25 A – Can hold Peptides 8-10 amino acids long

32. ~12 A

33. MHC I binding to peptide
Peptide backbone

34. Ag Site Class I
See figure 8-4, Kuby,
6th Edition, p. 195, right

35. Ag-Bind
See figure 8-9(a,b), Kuby, 6th Edition,
p. 199

37. See figure 8-9(c), Kuby,
6th Edition, p. 199
Ag in Site Class I

38. Class I MHC Space-Filling

39. H2K and Peptides
See figure 7-12,
Kuby 4th edition,
p. 184

40. MHC ClassI and Flu

41. MHC-Class I Polymorphisms
Kuby, 6th Edition
Figure 8-10(b)
p. 201
MHCIPoly
Positions of
Polymorphic Amino
Acid Residues in a
Human Class I
MHC Protein

42. Endogenous Ag processing… peptide generation
Proteins targeted for lysis combine w/ a small protein  ubiquitin
Ubiquitin-protein complex is degraded by a proteosome
Specific proteosomes generate peptides which can bind to MHC I

43. Proteosome
About 1/3 of intracellular proteolysis in mammalian cells is directed to
nascent proteins
- defective ribosomal products (DRiPs)
non-functional and potentially toxic proteins
proteins synthesized in excess (maintain protein homeostasis)
regulatory proteins
Only about 1% of the peptide pool is available to immune system

44. Immunoproteasome
P28 causes N-terminal tails of the -subunits to flip upwards, thereby facilitating
substrate entry and product exit.
The immunoproteasome does not replace the constitutive proteasome completely
The immunoproteasome has a considerably shorter half-life
The immunoproteasome has an altered cleavage site preference with a strong
preference to cleave behind residues that represent correct C-terminal anchors
for MHC I presentation.
PA28 does confer new cleavage site specificities, but enhances the frequency
of the usage of minor cleavage sites to provide more peptides for MHC presentation

45. 26S protease complex 20S proteasome twin 19S caps

46. TAP, transporter associated with antigen processing
structure:
TAP-1 and TAP-2
function:
transports small peptides (8-13 aa) to the ER

48. Endogenous Ag processing… transport to ER
Peptides from proteolysis bind to a “Transporter protein associated with Antigen Processing” (TAP)
TAP is a heterodimer which uses ATP to help transport peptides (8-13 amino acids) to lumen of ER
Usually basic amino COOH end of peptide chain

49. Trim-peptidases Cytosolic peptidases
Peptides produced by proteasomes are often to large for presentation (8-11 aa)
or for TAP transport (8-16 aa)
Several cytosolic and ER proteases are involved in trimming.
However, their major function is probably peptide degradation for amino acid recycling.
Cytosolic peptidases
Puromycin-sensitive aminopeptidase (PSA):
- metallopeptidase
- shown to both trim and destroy epitopes
Thimet oligopeptidase (TOP):
- metallopeptidase of the M3 family
- peptides of up to 15 aa are preferred substrates
- mainly involved in epitope destruction (down-regulation enhances presentation).

50. Cytosolic peptidases (cont.)
Leucine aminopeptidase (LAP):
- metallopeptidase
- peptides of less than 7 aa are preferred substrates
- mainly for aa recycling
Tripeptidyl protease II ((TPP II):
- cleaves peptides larger than 15 aa
- plays significant role in antigen presentation
- exopeptidase activity: removes blocks of 3 N-terminal amino acids
- endopeptidase activity: trypsin-like specificity
There are no carboxypeptidases in the cytosol
The proteasomes have to generate C-terminal anchor for MHCI binding

51. ER peptidases
ER aminopeptidase associated with antigen processing (ERAAP):
= ERAP1 (human)
- metallopeptidase of M1 family
- specific for large hydrophobic residues, such as Leu
- strong preference for substrates of 10 or more aa
ERAP2:
- 49% identical to ERAP1 by aa sequence
- specific for basic residues, such as Arg and Lys.

53. Endogenous Ag processing… peptide binding to MHC I
MHC I assembly occurs w/ the aid of chaperone proteins to promote folding (calnexin + MHC I α chain)
Tapasin + calreticulin brings TAP/ peptide close to MHC assembly
Allows MHC I to bind to peptides
MHC I-Ag exits ER to Golgi to plasma membrane

54. calnexin Structure Function
88kD integral ER membrane chaperone protein
Function
Binds to a nascent MHC class I a chain after release from a ribosome into the ER lumen so that the a chain will not leave the ER until it binds both a short peptide sequence and b2 microgobulin

55. Tapasin 48 kDa glycoprotein
stabilises TAP1/TAP2 which enhances peptide transport
bridges MHC class I to TAP (structural component)
facilitates peptide loading
stabilizes “empty” peptide-receptive MHC complexes
optimizes peptide repertoire (peptide editor)

56. Molecular chaperones: calnexin, calreticulin,tapasin

57. Assembly and stabilization of MHC I – Ag complex

59. Major Histocompatibility Complex Class I
Antigen is usually endogenous (e.g. viral proteins).
CD8+ cytotoxic T lymphocytes (CTLs) recognize antigen in association with class I MHC on APC.
MHC class I is expressed in all nucleated cells.

60. “Lost in action” or “the inefficiency of antigen presentation”
about 2 billion proteins per cell are expressed and turned over in 6h
about 100 million peptides per cell are generated in 1 minute
only a few hundred MHC I molecules are made in 1 minute
a large fraction of MHC I molecules fail to acquire a peptide
a peptide has an average in-vivo half-life of a few seconds
more than 99% of cytosolic peptides are destroyed before their
encounter TAP
An antigen has to be expressed at a minimum of 10,000 copies to be
presented by MHC class I

61. MHC CLASS II Expressed in Professional Antigen Presenting Cells-APCs
Dendritic cells, B-Cells, Macrophages

62. MHC-II
See figure 8-3, Kuby,
6th Edition, p. 194, right

63. Class II Molecules Antigen is usually extracellular.
CD4+ Helper T Lymphocytes recognize antigen in association with class II MHC gene product on APC.
Found only on “professional antigen presenting
cells”, such as dendritic cells, macrophages, B cells

64. Peptide binding Cleft

66. Endocytotic PROCESSING:
INVARIANT PROTEIN (Ii) is synthesized with the alpha and beta chains of MHC-II.
The invariant chain exists as a trimer and has attached to it three sets of alpha/beta chains.

75. ANTIGEN PRESENTATION

78. Antigen Presentation by Macrophage
T Cell

79. Numerous T Cells
Interacting with a
Single Macrophage

85. T-cell Receptor and MHC Class I Complex

87. SIGNAL TRANSDUCTION BY THE TCR
Stimulation of the TCR by antigen-MHC triggers the phosphorylation of tyrosine residues in the cytoplasmic domains of the CD3 chains of the receptor complex.
According to a widely accepted model of TCR signaling, Lck and Fyn are responsible for these initial phosphorylation events.
2/9/04

88. Immune-receptor-Tyrosine-based-Activation-Motif; ITAM