Notion of immunological “SELF”Skin graft transplantation compatibility

• Graft compatibility genetically determined

Extremely polymorphic trait -many alleles

Governed by genes of major histocompatibility complex (MHC) that encode MHC molecules, the principal targets of rejection

• Rejection by adaptive immune system T cell response

Differences in MHC molecules between individuals are central to determining “SELF”

How T cells recognize antigen

• The peptide is not directly identified by the TCR, but recognized in a form bound to a MHC molecule

• Each clonal T cell receptor (TCR) is specific for a particular sequence of amino acids in a peptide

• The peptide is generated from proteins in the peptide-presenting cell

Clonal recognition of antigen by T lymphocytes

The huge diversity of pathogen peptides to be recognized mandates a large number of different T cell clones, necessitating:

Clonal recognition of antigen by T lymphocytes

• A somatic recombination mechanism to generate the large number of clones

• A clonal selection process to identify the appropriate clones for the clonal repertoire

The selection process (Thymic “education”) has two stages

• First stage selects clones capable of recognizing self peptide in an individual’s own MHC molecules - positive selection

• Second stage eliminates overtly self reactive clones with high affinity for self peptide:MHC- negative selection

(Self-peptides are used as a surrogate for foreign peptides since there are few non-self peptides)

***

• The specificity of self/non-self peptide binding to MHC molecules determined by pockets that only bind certain amino acid side chains

• MHC genes are extremely polymorphic and alleles encode pockets with specificities for different amino acid side chains

The TCR is specific for both peptide and MHC- A complex ligand Polymorphic

residues of MHC

The TCR repertoire differs from individual to individual

The definition of immunologic self is made by selecting the clonal T cell repertoire on self-peptides bound to the individual’s particular allelic forms of MHC molecules

Immunologic self is both the collective

recognition specificity of an individual’s T cell

repertoire for peptides presented by the MHC

and the set of self-peptides and self-MHC

molecules that generated the repertoire

Non-self peptides from pathogens, etc., are bound to MHC molecules and recognized by T cells because their peptides are partial mimics of self

Many of the events in the clonal selection process are recapitulated during the clone’s response to peptide in an immune response

The immune response to pathogens

Polymorphic residues of MHC

Because of MHC polymorphisms we each likely recognize non-self antigens and pathogens differently from the next individual

Major selective advantages to the species

However because the adaptive immune system is patterned on self, it sets the stage for the development of autoimmune disease

Virus - or Pathogen - infected cell

Bacteria or components of an extracellular pathogen

that have been internalized

Any nucleated cell Macrophage/DC

T cells recognize peptide antigens from two main types of pathogens

B cell

A viral peptide on a cell signifies it is infected and should be killed, while a pathogen’s peptide on a phagocytic cell signifies the cell has ingested a foreign substance and must be helped to eliminate the pathogen

The adaptive immune system must make this distinction

Cytosolic Virusor Pathogen

Ingested Bacteria or Endocytic Pathogen

Extracellular Pathogenor Toxin

Peptide degraded in:

Peptides bind to:

Presented to:

Effect on presenting cell of T cell recognition:

Cytosol

MHC class I

CD8 T cells

Death of cell presenting the viral antigen

Endocytic vesicles

MHC class II

CD4 T cells

Provision of help to B cell for production of antibodies

Endocytic vesicles

MHC class II (or I)

CD4 T cells (or CD8)

Activation of cell to enhance pathogen killing

Challenge:

Any cell Macrophage/DC B cell

The immune system makes this distinction by loading and recognizing peptides in either class I or class II MHC

Presenting cell:

Structure of MHC molecules

What are the structural features of the MHC that determine peptide binding?

0 1 2 3 4m bp

Class II loci Class I loci

HLA-AHLA-BHLA-C

HLA-DRHLA-DQHLA-DP

Organization of the MHC

Ch 6 Human Leukocyte Antigens (HLA)

Two classes of MHC molecules

Structure of peptide-binding class I MHC domain

N

1

2

MHC Class I Domains

Peptide-binding domains

2m

The ligand for the CD8 T cell TCR

Class I Class II

The overall structure of class I and class II MHC is rather similar

Peptide

MHC class I molecule MHC class II molecule

2m

1 2

3

1 1

2 2

MHC class I and II molecules have homologous domain organization, but different chain structure

The Structure of MHC Molecules: MHC Class I

• Transmembrane and cytoplasmic portion

• Three globular domains, 1, 2 and 3, each ~90AA

• 2 microglobulin ~100AA, associates with the 3 domain, not MHC encoded

• The chain is~ 350 AA long

• 1 and 2 form the antigen-binding cleft

The Structure of MHC Molecules: MHC Class II

• Composed of two similar membrane spanning proteins, the -chain and chain both encoded within the MHC

• Each chain is made of two globular domains, each ~90AA, e.g. 1 and 2

• 1 and 1 domains form the antigen-binding cleft

At the genetic level the MHC class I and II system is complex!

Two levels of diversity:

Within individual: duplicated loci

Class I: HLA-A, HLA-B, HLA-C

Class II: HLA-DR, HLA-DQ, HLA-DP

Between individuals: hundreds of alleles for most of these loci

The MHC is by far the most genetically polymorphic region in the genome

The diversity of MHC class I and II genesAn evolutionary response to the structural diversity and mutation potential of microorganisms

Arises from two mechanisms:Duplication of a gene locus in an individual resulting in multiple loci, polygeny

Development of multiple alleles at a locus among individuals in the species, polyallelism

(isoforms or isomorphs)A A B

A1

A2

A3

A4

Alleles

A1

A2

A3

A4

B1

B2

B3

B4

(Allotypes)In different individuals

MHC alleles have a dominant mode of action because the molecules they encode have the ability to bind particular peptides

Corollary: if your MHC molecules cannot bind a peptide, you cannot make a T cell response against it

Duplicating a locus allows it to mutate and develop new peptide presenting specificities that operate in parallel with older ones

HLA-AHLA-BHLA-C

HLA-DRHLA-DQHLA-DP

However each duplication increases “self” and mandates more negative clonal selection during repertoire formation, reducing the size of the repertoire

Practical maximum is ~ three loci each for class I and class II

No limit on the number of alleles

Notion of immunological “SELF”Skin graft transplantation compatibility

• Graft compatibility genetically determined

Extremely polymorphic trait -many alleles

Governed by genes of major histocompatibility complex (MHC) that encode MHC molecules, the principal targets of rejection

• Rejection by adaptive immune system T cell response

Differences in MHC molecules between individuals are central to determining “SELF”

How T cells recognize antigen

• The peptide is not directly identified by the TCR, but recognized in a form bound to a MHC molecule

• Each clonal T cell receptor (TCR) is specific for a particular sequence of amino acids in a peptide

• The peptide is generated from proteins in the peptide-presenting cell

Clonal recognition of antigen by T lymphocytes

The huge diversity of pathogen peptides to be recognized mandates a large number of different T cell clones, necessitating:

Clonal recognition of antigen by T lymphocytes

• A somatic recombination mechanism to generate the large number of clones

• A clonal selection process to identify the appropriate clones for the clonal repertoire

The selection process (Thymic “education”) has two stages

• First stage selects clones capable of recognizing self peptide in an individual’s own MHC molecules - positive selection

• Second stage eliminates overtly self reactive clones with high affinity for self peptide:MHC- negative selection

(Self-peptides are used as a surrogate for foreign peptides since there are few non-self peptides)

***

• The specificity of self/non-self peptide binding to MHC molecules determined by pockets that only bind certain amino acid side chains

• MHC genes are extremely polymorphic and alleles encode pockets with specificities for different amino acid side chains

The TCR is specific for both peptide and MHC- A complex ligand Polymorphic

residues of MHC

The TCR repertoire differs from individual to individual

The definition of immunologic self is made by selecting the clonal T cell repertoire on self-peptides bound to the individual’s particular allelic forms of MHC molecules

How peptides bind to class I MHC molecules

How does polymorphism influence binding of different peptides and what is its immunologic significance?

Specific bound peptide always oriented in the same direction in the MHC

NH2COOH

Cluster of tyrosines recognize NH2

+ charged amino acids interact with negative COOH

How peptides bind

Class I MHC

Length of peptide is constrained to 8 or 9 amino acids

How peptides bind3

2

5 7

9

peptideN C

MHC class I

• Usually peptides are 8-9 amino acids in length• Always oriented with NH2 terminus to the left• Most often anchored by interactions of the side chains of their 2nd (P2) and 9th (P9) amino acids to MHC pockets that confers specificity for amino acids with similar physical properties, e.g. size, charge, hydrophobicity, etc.

Rules for binding to MHC class I molecules

TCR

AnchorAnchor

DifferentProteins yield different peptides that can bind to the same MHC molecule because they share anchor residues

Each different MHC Class I allotype has pockets that preferentially bind different amino acid side chains at positions P2 and P9

Y

Y

Y

L

L

I

Y=TyrL=LeuI=Ile

Pr 1

Pr 2

Pr 3

Only a few peptides from each protein will bind to a given MHC class I allotype

Similar pockets for peptide side chains, but peptides binding class II molecules vary in length 12-24 AA and are anchored in the middle

Peptide binding to class II MHC molecules

1

2

4

5

6

7 8

9

peptide

Protein 1

Protein 2

Protein 3

• Binding is highly stable with no peptide interchange on cell surface• Specificity for original loaded peptide is maintained because empty MHC molecule is very unstable

For both class I and class II molecules

How do cytosolic peptides from protein synthesized in virally infected cells get loaded on class I and not class II molecules to trigger killing by CD8T cells?

How do peptides from ingested proteins get loaded on class II and not class I molecules to elicit macrophage activation and B cell help?

How are proteins metabolized within the cell to yield peptides that bind to the MHC?

The endocytic and synthetic pathways are usually quite separate

endocytic

Synthetic

Class I peptide loading

Cytosolic peptides synthesized by virally infected cells get loaded on class I MHC

Peptides generated in the cytosol by the proteasome bind to MHC class I molecules while they are being synthesized within the ER

Top Side

The 26S proteasome is composed of the 20S proteasome core containing the catalytic chamber and two 19S regulatory complexes that bind and unfold ubiquitylated proteins

3 of the 7 subunits in each ring of the 20s core confer the proteolytic activity, 1, 2 and 5

Responsible for the proteolysis of polyubiquitylated proteins in basal protein turnover

Proteasome core catalytic chamber cross section showing a ring of 7 subunits

Three have proteolytic activity; 1, 2 and 5

Proteins are unfolded and pass through the active proteolytic sites

• Upregulates classic MHC I and II and TAP gene products• Upregulates the synthesis of three new proteasome subunits with different proteolytic activity that replace the three constitutively active proteolytic subunits, changing specificity of proteasome towards production of hydrophobic peptides that have greater binding affinity for MHC pockets

• An immunomodulatory cytokine produced by activated T helper lymphocytes, CD8 CTLs and natural killer (NK) cells

IFN- greatly enhances antigen processing and presentation

Generation of the immunoproteosome

3 immunosubunits LMP2 (i1), LMP7 (i5) and MECL-1 (i2) replace the constitutive 1, 5 and 2 subunits in the 20S core and change specificity to more hydrophobic peptides

Generation of peptides for class I MHC requires precise proteolysis because the overall peptide length is restricted to 8 or 9 residues

The 26S immunoproteasome system is used to get precise cuts at the C termini of the peptides

Other peptidases nibble back the N termini until the peptide fits exactly, e.g. the IFN--inducible cytosolic leucine aminopeptidase (LAP) cleaves a single residue from the N- terminus

The process has a relatively low efficiency, especially since the peptide production system is not coordinated with the peptide binding specificity of the individual’s MHC class I molecules

A proportion of newly synthesised proteins are defective ribosomal products (DRiPs) that are rapidly ubiquitylated and degraded by proteasomes

DRIPS are the major source for the generation of antigenic peptides

Almost no peptides presented by class I molecules are derived from “old” proteins

Endocytic

synthetic

Class II peptide loading pathway

Proteins endocytized by DC, B cell or macrophage are contained in endocytic vesicles

Class II presentation is centered in the vesicular system

Acidic endosomal proteases digest ingested proteins into peptides that will load MHC class II molecules

This process does not require the precise proteolysis needed in the class I system, since the peptide terminii are not constrained by MHC class II

A chaperone that complexes with MHC class II molecules during their synthesis in the endoplasmic reticulum has two critical actions

• Ii Blocks the class II peptide binding groove and prevents loading by peptides destined for class I molecules

• The recognition sequence on the Ii transmembrane portion targets the nascent MHC II molecule from the synthetic compartment to the acidic endosomal compartment to join with the degraded ingested exogenous peptides

Invariant chain (Ii)

Ii is degraded to CLIP (Class II-associated invariant chain peptide) by specific endosomal acidic cysteine proteases (cathepsins)

• HLA-DM, an ancient but non-classical class II molecule catalyzes the release of CLIP and the binding of high affinity peptides via interaction of peptide amino acid side-chains with MHC pockets

CLIP is only bound to the MHC groove by its peptide backbone and its side chains do not engage the pockets

• Without Ii the MHC class II molecule traffics to the cell membrane