Antigen Processing & Presentation

 Endogenous Ags: Ags synthesized within cells, including self & unself protein – class I MHC
molecules. eg. Normal cellular proteins, tumor proteins, viral & bacterial proteins produced
within infected cells

 Exogenous Ags: Ags from outside the cells, including self & unself protein -- class II MHC
molecules.

 Antigen processing: Conversion of native proteins to peptides which can combine with MHC
molecules.

 Antigen presentation: Course of formation & display of peptide-MHC complexes on the surface
of APCs & recognition by T cells.

 Class I MHC mol - bind peptides derived from endogenous Ags

 that have been processed within the cytoplasm of cell

 Class II MHC mol bind peptides derived from exogenous Ags

 that are internalized by phagocytosis or endocytosis & processed within endocytic pathway.

Self MHC Restriction

• CD4+ & CD8 T+ cells can recognize Ags only when it is processed with a self-MHC mol on the
membrane of APCs

• This attribute - called self-MHC restriction, distinguishes recognition of Ag by T cells from that by
B cells

Antigen Presenting Cells (APCs)

• Cells that display peptides associated with MHC class II molecules to CD4+ T H cells

• Distinguishing features

- ability to express class II MHC mol

- deliver a co-stimulatory signal

Professional & Non-professional APCs

Professional APCs

• DC

• Mφ
• B cells

• Non-professional APCs

• Fibroblasts (skin)

• Thymic epithelial cells

• Thyroid epithelial cells

• Glial cells (brain)

• Pancreatic beta cells

• Vascular endothelial cells

• Non-professional APC:

• - does not constitutively express MHC class II (required for interaction with naive T cells)

• - expressed only upon stimulation of non-professional APC by certain cytokines such as IFN-γ.

• DC – (most effective ) - Constitutively express high level of MHC II mol & co-stimulatory activity

• Mφs – must be activated by phagocytosis of pathogen à express MHC II & B7

• B cells – constitutively express MHC II, must be activated to express B7

• Immune system uses two different pathways to eliminate intracellular & extracellular Ags

• Extracellular Ags are eliminated by secreted Abs

• Exogenous Ags - taken up by endocytosis are processed in endocytic pathway & presented on
the membrane with class II MHC molecules

• Intracellular Ags are eliminated by CTLs

• Endogenous antigens (those generated within cell) are processed in cytosolic pathway &
presented on the membrane with class I MHC molecules
 Class Ⅱ MHC pathway

1. Capture of exogenous Ag

2. Processing of Ag

3. Synthesis & transportation of class Ⅱ MHC molecules

4. Peptide loading of classⅡ MHC molecules

5. Presenting to CD4+T cells

Ag capture & internalization

 APCs can internalize Ag by phagocytosis, endocytosis or both.

 Mφs internalize Ag by both processes, whereas most other APCs are not phagocytic or poorly
phagocytic - therefore internalize exogenous Ag only by endocytosis (either receptor-mediated
endocytosis or pinocytosis)

 B cells, for eg, internalize Ag effectively by receptor-mediated endocytosis using Ag-specific

Generation of Peptides in Endocytic Vesicles

• Once an Ag is internalized, it is degraded into peptides within compartments of the endocytic

• This pathway - three increasingly acidic compartments:

– late endosomes, or endolysosomes (pH 5.0–6.0);

– lysosomes (pH 4.5–5.0).

– Internalized Ag moves from early to late endosomes & finally to lysosomes,

• Within compartments, Ag is degraded into oligopeptides of about 13–18 residues, which bind to
class II MHC mols.

• Because hydrolytic enzymes are optimally active under acidic conditions (low pH), Ag processing
can be inhibited by chemical agents that increase pH of the compartments (e.g., chloroquine).

Invariant Chain Guides Transport of Class II MHC Molecules to Endocytic Vesicles

• APCs express both class I & class II MHC molecules, some mechanism must exist to prevent class
II MHC molecules from binding to same set of antigenic peptides as class I mols.

• When class II MHC molecule are synthesized within RER, three pairs of class II chains associate
with a preassembled trimer of a protein called invariant chain (Ii, CD74).

• This trimeric protein interacts with peptide-binding cleft of class II molecules, preventing any
endogenously derived peptides from binding to the cleft while class II molecule is within RER.

• Invariant chain – also involved in folding of class II chains, their exit from the RER & subsequent
routing of class II mols to endocytic processing pathway from trans-Golgi network.

Peptides Assemble with Class II MHC Mols by displacing CLIP

• Class II MHC–invariant chain complexes - transported from RER (where they are formed),
through Golgi complex & trans-Golgi network, then through endocytic pathway, moving from
early to late endosomes & finally to lysosomes.

• As proteolytic activity increases in each successive compartment, invariant chain is gradually

• However, a short fragment of invariant chain - CLIP (class II–associated invariant chain peptide)
remains bound to class II molecule after the invariant chain has been cleaved within endosomal
compartment.

• CLIP physically occupies the peptide-binding groove of class II MHC molecule, presumably
preventing any premature binding of antigenic peptide.

HLA-DM
• Nonclassical class II MHC mol – HLA-DM plays a role in removal of CLIP & in subsequent loading
of class II molecules with antigenic peptides.

• HLA-DM - required to catalyze exchange of CLIP with antigenic peptide.

• Reaction bw HLA-DM & MHC class II-CLIP complex facilitating exchange of CLIP for another
peptide

• MHC class II genes encoding HLA-DM - identified in mouse & rabbit, indicating HLA-DM - widely
conserved among mammalian species.

• Like other class II MHC molecules - HLA-DM is heterodimer of α & β chains.

• Unlike other class II molecules - HLA-DM not polymorphic & not expressed at cell membrane but
found predominantly within endosomal compartment.

• DMα & DMβ genes - located near TAP & LMP genes in MHC complex of humans.
 The Cytosolic Pathway

 Endogenous Antigens

 Degraded within cytoplasm to peptides

 Associate with class I MHC molecule

Steps of ClassⅠMHC pathway

1. Processing of endogenous Ag

2. Transporting of antigen peptide into ER

3. Peptide loading of class Ⅰ MHC molecules

4. Presenting to CD8+T cells

Processing of endogenous Ag

 Endogenous Ags: denatured, misfolded, abnormal proteins

 Intracellular proteins – degraded into short peptides by cytosolic proteolytic system

 Proteins targeted for proteolysis – have small protein ubiquitin attached to them

 Ubiquitin-protein conjugates – degraded by multifunctional protease complex – proteasome

 Peptide Generation by Proteasomes

 Proteasome – large, cylindrical particle consisting of four rings of protein subunits with central
channel of 10-50 Å

 Degradation of ubiquitin-protein complex – central hollow of proteasome

 Prevent lysis of other proteins within the cytoplasm

 Protein to be degraded – covalently linked to ubiquitin

 links several ubiquitin mols to lysine aa near amino terminus

 require ATP

 Till now: general pathway of protein degradation

 Immune system modify proteasome

 By addition of other subunits to proteasome

 LMP2, LMP7 and LMP10

 LMP10 = MECL-1

 Induced by increased levels of IFN-γ

 Peptidase activity of proteasome containing LMP2, LMP7 and MECL1 – generate peptide that
bind MHC I mol

 Transporting of antigen peptide into ER

 Transporter proteins:

 TAP (transporter associated with antigen processing)

 Membrane spanning heterodimer

 Consisting of TAP1 and TAP2

 ATP dependent transporter of aa, sugars, ions & peptides

One hydrophobic domain – project into lumen of RER

One ATP-binding domain – projects into cytosol

Assembly of peptides with class I MHC mol\

 TAP transport - Peptides 8-13 amino acids

 Optimal peptide length for MHC class I mol

 Like other proteins α chain and β2-microglobulin of MHC class I – synthesized in RER

 Molecular chaperones – facilitate folding of polypeptides

 Mol chaperon first involved in MHC I assembly is calnexin

 Calnexin associate with free class I α chain & promote its folding

 When β2 microglobulin binds to α chain, calnexin is released

 Class I mol associates with chaperonin – calreticulin & tapasin

 Tapasin (TAP-associated protein)

 Brings TAP transporter into proximity with class I mol

 Allow it to acquire Ag peptide

 Antigen peptide-class Ⅰ MHC molecuels presented on cell membrane by exocytosis