HLA and Antigen Processing 9/4

Question 1

HLA

Answer 1

Function as antigen-presenting structures to T-cells

T-cells DO NOT recognize antigens in free or soluble forms

Recognize portions of protein antigens (peptides) associated with HLA

Differences in HLA molecules expressed by an individual will influence the repertoire of antigens to which T-cells can respond

Question 2

HLA Genes

Answer 2

Tightly linked cluster of genes that are highly polymorphic.

• HLA has unprecedented extent of polymorphism
• More than 150 separate alleles have been identified within the HLA
• Many alternative versions of each HLA gene

HLA complex genes: chromosome #6

• HLA genes are divided into three distinct classes: class I (HLA-A,B,C), class II (D region) and class III (C4B, C4A, Bf, C2, TNF)

Question 3

HLA Haplotype

Answer 3

• The total set of HLA alleles are present on each chromosome. The set of alleles on each chromosome is called an HLA haplotype.
• Encode protein antigens central for immune system to discriminate between self and non-self.
• Most human are heterozygous and have two HLA haplotypes
• one from Mom and one from Dad
• Both haplotypes expressed simultaneously (co-dominate expression) – no allelic exclusion here - Generating more diversity
• Transplantation
• Want the best match possible between recipient and donor for both class I and class II genes

Question 4

Class I HLA

Answer 4

** Function1: Presents the antigen to CD8+ cytotoxic T-cells (CTL):

**Function 2: Also functions as inhibitory receptor for NK cells: All nucleated cells express Class I, so that when the NK cell bumps to check, it is inhibited by inhibitory receptor ligating with class I

• Structure:
• membrane bound glycoprotein, expressed on all nucleated cells
• Four extracellular globular domains
• Heterodimer of two proteins: Alpha chain, Beta2-microglobulin

–>Alpha Chain:

• encoded by HLA class locus
• forms three of four globular domains (alpha 1,2,3)\
• alpha 1 and alpha 2 form peptide binding cleft
• alpha 3 is not polymorphic, it is where CD8 binds - same for everyone

–>Beta-2 Microglobulin:

• non-HLA encoded (same for everyone)
• forms fourth domain
• associates non-covalently with the a3 domain
• Class I Genes:
• Encoded by three separate gene regions in the HLA locus (HLA-A, B, C)
• Key features:
• All alleles of Class I can be expressed at the same time on each cell (A/B/C from mom and dad = 6 different HLA’s)
• Each one has a slightly different shape and presents a different set of peptides

Question 5

Peptide binding groove of HLA 1

Answer 5

• area between the alpha1 and alpha2 domains where the peptides are bound and presented on the surface of the cells
• site with the greatest polymorphism
• binds peptides about 8-10 aa’s in length (closed end limit size)
• the conformation of this groove dictates what peptides can bind: each allele of Class I HLA has a different range of peptides that can bind in the groove

Question 6

Class I HLA synthesis

Answer 6

• alpha chain is translated into ER as glycoprotein
• In the ER: alpha chains interact with Beta 2 microglobin, and Class I associates with peptides
• Class I HLA/Peptide complex is tranported to the cell surface via the normal trafficking of glycoproteins through ER and golgi

Question 7

Class II HLA

Answer 7

Function: Primarily located on APC’s: macrophages, dendritic cells, and B cells: present antigen to CD4+ T Cell (Th cell)

• Genes:
• HLA-D region: 3 sets of genes: PQR (HLA-DP, DQ, DR)
• alph and beta chains
• membrane bound glycoproteins
• structure:
• composed of two proteins: alph and beta chain - both encoded by HLA-D region
• four globular domains: similar to Class I
• alph and beta chains are strongly associated- NOT covalently linked
• Key features:
• all alleles from alpha and beta chains are expressed on cell (6 total)
• any alpha chain allele may associate with any beta chain allele –> adds to diversity of the peptide binding groove
• ***Greater range of peptides can bind to the Class II HLA

Question 8

Peptide Binding Groove of Class II HLA

Answer 8

• formed by the alpha1/beta2 globular domains (have greatest polymorphism)
• binds peptides between 13-18 aa’s
• open ends allow larger peptides to bind (hang out like a long hotdog)
• conformation of this groove dictates what peptides can bind
• each allele of Class II HLA has a different range of peptides that can bind in the groove

Question 9

Class II HLA synthesis

Answer 9

• alpha/beta chains synthesized in the ER
• they interact with a third protein - “invariant chain” protein
• the invariant chain binds the alpha and beta chains because they are noncovalently associated
• blocks the peptide binding groove
• directs the transport of the Class II molecule to an endocytic compartment
• in the endocytic compartment
• invariant chain is degraded
• free peptides bind the groove
• classIi molecules bind peptides that have entered the cell via endocytosis (the protein antigens cam from outside of the cell)
• peptide bonding is non-specific aside form the length of the peptide that will fit it
• Class II HLA/peptide comlex is then transported to the cell surface

Question 10

Peptide binding

Answer 10

• There are pockets in the floors of the peptide-binding clefts. The side chains of the amino acids on the antigen peptides fit into these pockets and anchor the peptides in the cleft. The rest of the peptide contains some residues that bow upwards and are recognized by the Ag receptors on T cells.
• peptide bonding is not dependent on shape or specific AA’s, it is only dependent upon SIZE. Not speicific binding!
• Key Features:
• saturatable binding, low affinity interaction
• slow “on rate”
• very slow “off rate” - allows peptide HLA complexes to persist for interaction with T-cells
• only one peptide binds to a molecule of HLA at a time
• However, the same HLA molecule can bind a variety of multiple peptides

Question 11

H-Y

Answer 11

• minor HLA
• Ag coded on Y chromosome
• associated with acute rejection of male grafts in female recipients

Question 12

HLA Restriction

Answer 12

The ability of T cells to recognize antigens when associated with the organism’s own HLA haplotype, providing a dual recognition system critical to T-cell function.

What it means:

• CD4+T cells only recognize Ag bound to Class II HLA
• CD8+T cells only recognize Ag bound to Class I HLA

Question 13

APC’s

Answer 13

• Antigen presenting cells: convert proteins to peptides for display (presentation) on class II HLA
• present to CD4+ T cells

Three main types:

• macrophages, dendritic cells (most efficient), B cells
• sometimes other cells express Class II HLA (like thymic epithelial cells)

Question 14

Dendritic Cells

Answer 14

• professional APC = most effective, because they produce co-stimulatory molecules
• pinocytose Ag and process it for presentation with HLA
• Homing to T cell rich areas in nodes and in spleen
• Activate naive CD4+, and CD8+ T cells

Question 15

Macrophages

Answer 15

• phagocytose/pinocytose Ags
• not as effective as DC’s at activating naive T cells, but very good at activating memory T cells

Question 16

B cells

Answer 16

• bind soluble Ag via sIgs
• ingest via pinocytosis
• bind Ag with high affinity and thus are effective when Ag levels are low
• not as effictve as DC’s or macrophages in presentin Ag to naive T’s
• Very effective at presenting to memory T cells

Question 17

Capture of Ag’s

Answer 17

Microbes enter the body

Phagocytosed or pinocytosed by APCs

Lose adhesive markers and upregulate CCR7:

• movement to lymphatic vessicles

Increase expression of HLAI/II (both classes), B7

• B7 is costimulatory molecule for T cells
• lymph nodes filter Ag’s coming from periphery
• spleen filters Ag’s coming from blood (blood born Ags are captured by APCs in the spleen)

Question 18

Two Processing Pathways

Answer 18

Function of:

• Chemical nature of the fragment
• Density of protein
• Specific HLA & its binding site
• Self versus non-self

Each pathway stimulates the T cell population most effective against specific Ag

• Intracellular pathogens and self: Class I HLA
• Extracellular pathogens: Class II HLA

Question 19

Class II HLA Pathway

Answer 19

1. Exogenous proteins are ingested and degraded in APC
2. Procesing of internalized proteins in endosomal/lysosomal vesicles
3. biosynthesis and transport of class II MHC (alpha and beta and invariant chains are synthesized) in the ER
4. MHC II is transported through golgi
5. Association of processed peptides with Class II MHC molecules in vesicles (Peptide is not loaded until late endosomes)
6. Expression of peptide-MHCII complexes on cell surface

Question 20

Functions of Class II associated invariant chains and HLA-DM in the CLass II HLA pathway:

Answer 20

Ii occupies the peptide-binding cleft, promotes folding, assembly, trafficking, and place holder

Ii is degraded to CLIP in by lysomal enzymes

HLA-DM acts as a peptide exchanger, facilitating the removal of CLIP and the addition of peptides to Class II HLA

Unbound HLA molecules are not displayed

Steps of binding:

1. synthesis of class II MHC in ER
2. Transport of class II and Ii to vesicle
3. binding of processed peptides to class II
4. transport of class II peptide complex to cell surface
5. expression on cell surface

Question 21

Class I HLA Pathway

Answer 21

Cytosolic Antigens

Proteasome: found in cytoplasm of most cells, degrades damaged proteins, proteins are targeted by ubiquitin

TAP: transports peptides from cytosol to interior of ER, peptide is trimmed and load into Class I molecule

Steps of the pathway:

1. production of proteins in cytosol (such as viral proteins)
2. proteolytic degredation of proteins by proteosome
3. transport of peptides from cytosol to ER via TAP
4. assembly of peptide class I complexes in ER
5. Surface expression of peptide-class I complexes

Question 22

Outcomes of Ag presentation

Answer 22

• Dendritic cells uptake Ag, and via costimulator B7 and CD28 they associate with Naive T cell via the presentation of Ag on class II HLA presentation to naïve T cell
• in synapse IL2 is released to further stimulate T cell
• the response is Naive T cell activation: clonal expansion and differentiation into effector T cells
• Macrophage uptakes antigen and presents it via Class II HLA to effector T cell
• provided costimulatory cytokines allowing to better kill
• the response is effector T cell activation, and the activation of macrophages (cell-mediated immunity) to kill the microbe
• B cell binds the Ag via Class II HLA presentation to effector T cell
• IL4 – increased antibody production from B cells
• the response is effector T cell activatoin and B cell activation and antibody production (humoral immunity)

Question 23

Cross Presentation

Answer 23

• Often seen with virally infected cells
• the DC ingest virally infected cells and display the Ag’s to CTL’s using both HLA classes

*** Look back at this slide: 34

• The term cross-presentation denotes the ability of certain antigen-presenting cells to take up, process and present extracellular antigens with MHC class I molecules to CD8 T cells (cytotoxic T cells)
• Cross-presentation is of particular importance, because it allows for the presentation of exogenous antigens that are presented by MHC II of infected dendritic cells to be presented by MHC I without requiring infection of dendritic cells and the subsequent functional impairment of dendritic cells using MHC I antigen proteasomal processing mechanism (coupled with the process of cytosolic diversion away from the processes of MHC II presentation), which is usually used for the presentation of endogenous antigens processed following infection

Question 24

Ankylosing spondylitis

Answer 24

• Inflammation of the spine
• Over 88% of individuals express the HLA-B27 allele
• Each allele has limited number of peptides it can present
• possible that the HLA-B27 allele can not bind a critical antigenic peptide
• present a critical antigenic peptide against the agent causing the disease

Question 25

Other HLA associated Diseases

Answer 25

Rheumatic fever

• Sequelae of Streptococcus pyogenes infection
• Generation of antibodies against the streptococci (cross react with cardiac tissue)
• Patients who have the HLA-DR4 allele
• more prone to develop Rheumatic fever

Other HLA associations

Sjögren’s Syndrome

• associated with HLA-DR3
• Defect in salivation and lacrimation

Insulin-dependant diabetes mellitus

• associated with HLA-DQw8

Psoriasis

• Associated with HLA-B3

Question 26

Processing Defects diseases

Answer 26

Human Neuroblastoma:

• Multiple defects of the antigen-processing machinery components in human neuroblastoma

Renal Cell Carcinoma

• Class I Antigen-processing defects in renal cell carcinoma
• Transporter associated with antigen processing is down regulated (TAP)

Question 27

summary slide

Answer 27

HLA-A, HLA-B, and HLA-C gene loci encode Class I HLA molecules.

HLA-DP, HLA-DQ, AND HLA-DR gene loci encode Class II HLA molecules.

An individual’s HLA haplotype affects susceptibility to disease.

T cells recognize peptide fragments that have been processed and become bound to class I or II HLA molecules.

Class I HLA molecules associate with endogenously synthesized peptides, binding to peptides produced by degradation of the cells’ internal molecules. This type of antigen processing is carried out by proteasomes and TAP (which take the fragments to the).

Class II HLA molecules bind to peptides produced following the breakdown of proteins that the cell has endocytosed. The peptides produced by degradation of these external antigens are loaded onto Class II HLA molecules.

Cross-presentation allows APCs to acquire antigens from infected cells. Cross-presentation, allows the display of exogenous antigens by Class I HLA molecules.

Antigen presentation affects the subsequent course of an immune response. The immune system responds to clues that an infection has taken place before responding strongly to antigens.

CD4 binds to Class II HLA and CD8 to Class I HLA molecules. These interactions increase the affinity of T cell binding to the appropriate HLA–antigen complex and bring kinases to the TCR complex.