1. Antigen Presentation And Processing
W. Robert Fleischmann, Ph.D.
Department of Urologic Surgery
University of Minnesota Medical School
(612)

2. Objectives
To understand the methods by which endogenous and exogenous antigens are processed and presented to the immune system on Class I and Class II MHC molecules.

3. Overview of Ag-Presentation
B cells have antibodies on their surface that bind to epitopes on native antigens
However, most T cells cannot interact with native antigens.
Antigens must first be processed and presented to T cells in the context being bound to MHC molecules.
T cell receptors on the surface of T cells recognize the antigenic peptides bound to MHC molecules.
With appropriate co-stimulation and cytokine production, the T cell is activated.
Unless T cell activation occurs, adaptive immunity does not develop.

4. Role of Ag-Presenting Cells
Processing of antigen is required for recognition of an antigen by T cells.
Most cells in the body can present antigen with Class I MHC molecules.
This includes the presentation of foreign antigen and self-antigens.
CD8+ cytotoxic T cells recognize antigen bound to Class I.
Professional antigen-presenting cells present antigen with Class II MHC molecules.
CD4+ helper T cells recognize antigen bound to Class II.

5. Professional Ag-Presenting Cells
Dendritic cells are the most effective
Immature dendritic cells in peripheral tissues express low levels of Class II molecules. These cells take up and process antigen, then move to lymph nodes.
Mature dendritic cells in lymphoid tissues express high levels of Class II molecules. These cells are the primary presenters of antigen.
Constitutively express B7 and other costimulatory molecules
Present peptides, viral antigens, and allergens
Macrophages
Must be activated by phagocytosis of bacteria and by cytokines to express Class II molecules
Must be activated to express costimulatory molecules
Present particulate antigens: intracellular and extracellular pathogens
B cells
Constitutively express class II MHC molecules
Must be activated by antigen binding to antibody before they express costimulatory molecules
Present soluble antigens, toxins, viruses

6. Characteristics of Ag-Presenting Cells
B7 is needed to activate naïve T cells.

7. Professional Ag-Presenting Cells
Class II MHC Expression
B7 Costimulation Factor Expression
Antigens Presented
Immature Dendritic Cells
Constitutively express low levels
Constitutive expressed
Present peptides, viral antigens, allergens
Mature Dendritic Cells
Constitutively express high levels
Constitutively expressed
Macrophages
Must be activated by phagocytosis to express
Must be activated to express
Present particulate antigen, intracellular and extracellular pathogens
B Cells
Present soluble antigens, toxins, viruses

8. Different Types of Dendritic Cells
Lymphoid origin?
Kuby
Suppressive activity.
Secretes IL-10 and IFN-
Secretes IL-12

9. Antigen-Processing and Presentation
There are different antigen processing and presentation pathways for different MHC molecules.
Cytosolic Pathway: Presentation of antigen on Class I molecules requires intracellular protein synthesis of the endogenous antigen.
Endocytic Pathway: Presentation of antigen on Class II molecules requires the endocytic uptake of exogenous antigen.

10. Cytosolic Pathway
This pathway is used for presentation of endogenous antigens.
Endogenous proteins are constantly being synthesized and degraded.
Some of the rapidly degraded proteins are defective ribosomal products (DRiPs) that are synthesized incorrectly.
Many are degraded to amino acids but some persist in the cytosol as peptides.
Some of these peptides are sampled by the immune system and presented on the cell surface bound to MHC Class I molecules.

11. Cytosolic Degradation Pathways
Degradation of misfolded proteins is mediated by 20S proteasome.
Degradation of correctly folded proteins that are targeted for degradation by ubiquitin is mediated by 26S proteasome (20S proteasome plus 19S regulatory component that may attach to either end of the 20S proteasome).

12. Cytosolic Degradation Pathways
20S proteasome is composed of 28 polypeptide subunits arranged in 4 stacked rings of 7 subunits each.
The outer two stacked rings are composed of 7 different  subunits. The inner two stacked rings are composed of 7 different  subunits, 3 of which are proteases.
Enzymatic cleavage of proteins is thought to occur within the central tube.

13. Cytosolic Degradation Pathways
26S proteasome is composed of 20S proteasome plus two 19S regulatory peptides that associate with each end.

14. Cytosolic Degradation Pathways
20 S immunoproteasome is composed of 20S proteasome but with 3 of the  subunits substituted with new IFN--stimulated  subunits.
Immunoproteasome shows bias towards increased cleavage after hydrophobic amino acids and reduced cleavage after acidic amino acids. This makes these peptides favored by TAP and Class I molecules.

15. Peptide Transport to the RER
Peptides from the cytosolic pathway are transported from the cytosol to the rough endoplasmic reticulum (RER) where Class I molecules are synthesized.
The peptides bind to a transporter protein heterocomplex (TAP1 and TAP2) that extends across the RER membrane and are held for a period of time on the lumen side before being released into the lumen.
TAP = transporter associated with antigen processing

16. TAP Function TAP1 and TAP2 form a functional dimer.
Peptides move from the
proteasome to TAP dimer.
Peptide is moved through
endoplasmic reticulum by
passage through a tube in
the TAP dimer.
The peptide emerges on the
lumen side of the ER and is
ready to be attached to MHC
Class I.

17. TAP Function TAP complex has an affinity for
peptides of 8-16 aa. This is close to the optimal Class I binding size of 9 aa.
Final trimming by an endoplasmic reticulum aminopeptidase (ERAP1) occurs in the RER lumen. ERAP1 will degrade down to 8 aa. Peptides too small for Class I binding are degraded by ERAP2.
TAP complex has an affinity for peptides with a hydrophobic or basic carboxyl-terminal aa, the preferred anchor residue for MHC Class I.
Thus, TAP transports peptides that have an affinity for Class I.

18. Assembly of MHC Class I Molecules
MHC Class I  chain is synthesized on the RER.
It becomes associated with the chaperone protein calnexin that assists in proper folding.
Class I  chain then becomes associated with the 2 microglobulin chain, releasing calnexin.
ERp57, tapasin, and calreticulin become associated with the Class I complex.
ERp57 (endoplasmic reticulum protein 57)
Tapasin (TAP-associated protein) brings the Class I complex close to TAP transporter)
Calreticulin (chaperone protein)
The antigenic peptide is added, releasing the other factors.
Insertion of the antigenic peptide into Class I groove is a signal to transit the Class I molecule to the outer surface of the cell membrane.

19. Peptides Associated with Class I
There are an excess of Class I molecules in the lumen of the RER. Thus, appropriate peptides are rapidly bound to Class I molecules.
Peptides that pass through TAP and that are held for binding to Class I molecules have two unique characteristics.
They are 8-10 aa in length, most commonly 9 aa.
The contain specific amino acid residues that appear to be important for binding to the peptide cleft.
The carboxyterminal anchor is generally a hydrophobic aa (leucine, isoleucine, valine) but is sometimes a charged aa.
There is another anchor at the second or third aa from the aminoterminus that is also generally a hydrophobic aa.
Peptides unprotected by TAP are degraded.

20. Significance of Peptides Presented on Class I
Degradation and presentation of peptides on Class I permits the sampling of proteins that are synthesized in a cell.
Defective ribosomal products or DRiPs are proteins that are synthesized incorrectly.
DRiPs are rapidly degraded and presented on the cell surface bound to MHC Class I molecules
This permits recognition and killing of cells that have aberrant DNA and thus produce aberrant proteins.
Virus-infected cells contain a distinct 20S proteasome.
This 20S proteasome is induced by IFN- and TNF-.
The proteasome degrades and presents viral proteins on the cell surface bound to MHC Class I molecules.
This allows recognition and killing of cells that are virus infected.

21. TAP Deficiency
Some individuals have been identified who have TAP deficiency.
Cannot express Class I molecules and their antigenic peptides.
Cannot defend against bacteria
perhaps because of deficiency of IFN- activation of Ms
Overactive NK cells
NK cells monitor what cells to kill or not to kill by the density of Class I molecules on their surface
Develop necrotizing granulomatous lesions on skin of face and extremities.
Can be deforming
Believed to be the result of overactive NK cells

22. Endocytic Degradation Pathway
Exogenous antigens are processed through the endocytic degradation pathway.
The antigens are internalized into antigen presenting cells by phagocytosis or endocytosis or both.
Ms and dendritic cells internalize by phagocytosis
B cells internalize by receptor-mediated endocytosis
The internalized antigens are degraded in phagolysosomes or endosomes.
The antigenic peptides are associated with Class II MHC and expressed on the cell surface.

23. Schematic of Antigen Processing
The endocytic processing pathway moves antigens through 3 compartments with increasing acidity and various hydrolytic enzymes.
1-3 hrs is required for antigen to be processed and presented via the endocytic pathway.
Antibody-bound antigen is internalized in early endosomes (pH ), then late endosomes (pH ), and finally lysosomes (pH ).
Somewhere in this progression, the antibody is separated from the antigen and recycled to the cell surface.
Fusion of the late endosome with the lysosome exposes the antigen to more than 40 different proteases, nucleases, glycosidases, lipases, phospholipases, and phosphatases.
Antigen is degraded to oligopeptides of aa.
The oligopeptides are then bound to MHC Class II molecules which blocks further enzymatic digestion.
The Class II molecules are transported to the cell surface.

24. Role of the Invariant Chain
Once exogenous antigens have been degraded to peptides they need to be combined with MHC Class II molecules.
MHC Class II molecules are synthesized on the RER, trimerized and combined with a trimer of membrane bound invariant chains (Ii, CD74).
The invariant chain appears to have several functions.
Assists in folding of the Class II  and  chains.
Binds to the peptide-presenting site of the Class II molecules.
Assists in the transport of Class II trimers from the RER to the Golgi and from the Golgi to cytoplasmic vesicles via signals in its cytoplasmic tail.

25. Assembly of Class II Molecules
The MHC Class II molecules bound to invariant chain move from the Golgi through the endocytic pathway.
With this movement, proteolytic cleavage gradually digests the invariant chain, leaving a short fragment (CLIP) bound to the antigen presenting site on the Class II molecule. (CLIP = Class II-associated invariant chain peptide).
HLA-DM and HLA-DO are nonclassical and nonpolymorphic class II molecules
HLA-DM is encoded in the same region of the genome as TAP genes.
Clip bound to Class II

26. Assembly of Class II Molecules
A nonclassical class II MHC molecule, HLA-DM) is required to catalyze the exchange of antigenic peptide for CLIP.
Another nonclassical class II MHC molecule, HLA-DO regulates the effect of HLA-DM.
HLA-DO is expressed only on B cells and in the thymus.
Unlike other class II molecules, it is not induced by IFN-.
HLA-DO binds to HLA-DM, except in very acidic conditions. This could favor expression of peptides that pass through lysosomes in B cells.
Once peptide/MHC is placed on the cell surface, the binding is exceedingly tight and the peptide does not come off under physiological conditions.
HLA-DM and HLA-DO are nonclassical and nonpolymorphic class II molecules
HLA-DM is encoded in the same region of the genome as TAP genes.

27. Summary of
Antigen Processing

28. A Couple of Special Cases of Antigen Presentation

29. Cross-Presentation of Exogenous Ag
Sometimes APC present exogenous antigens bound to Class I molecules.
This is not well understood but is believed to occur in the lumen of the RER.
This may provide a selective advantage in that it would allow dendritic cells to phagocytose viruses and present viral antigens with Class I.
This causes the generation and activation of cytotoxic T cells that can kill virus-infected cells prior to the general spread of the infection.

30. Presentation of Nonpeptide Ags by CD1
Our immune system can respond to antigens that are not peptide antigens.
Glycolipids
Polysaccharides
T cells bearing  TCR rather than  TCR also express the CD1 family of nonclassical Class I molecules.
T cells with  TCR are believed to be more ancient than T cells with 
5 genes (CD1A-CD1E) encode CD1a-CD1e molecules.
CD1 molecules have structures very similar to Class I molecules and associate with 2 microglobulin.
For example, CD1 present glycolipids and mycolic acid from various Mycobacterium species.
Loading Ag onto CD1 appears to occur via a third, yet unidentified pathway.
CD1 molecules present to cytotoxic T cells as well as to NK cells (part of innate immunity).
CD Class I Overlap