• Pete Sieling• Phone: 825-6964• email: psieling@mednet.ucla.edu• Office: 52-127 CHS

• Reading for Wednesday (4-20) and Friday (4-22) lectures: Chapter 5 (pp169-198), Janeway et al. 6th edition.

M261ANTIGEN PRESENTATION/MHC CLASS II

ANTIGEN PRESENTATIONMHC CLASS II

• Importance of dendritic cells to T cell activation.• Compare and contrast MHC I and MHC II peptide

binding.• MHC class II antigen processing and

presentation pathway.• Mechanisms of immune evasion.• Cross-presentation.• CD1 antigen presentation pathway.• Translational therapies targeting MHC antigen

presentation pathways.

CD4 T-CELL

TCR CD4

CD3

9 aa peptide15 aa

peptide

CD8 T-CELL

CD8

TCR

CD3

ANTIGEN PRESENTING CELL

MHCCLASS II

11

2 2

MOLECULES OF T LYMPHOCYTE RECOGNITION

CLASS IMHC 2m

2 1

3

ANTIGEN PRESENTING CELL

WHAT DISTINGUISHES MHC CLASS I FROM MHC CLASS II ANTIGEN PRESENTATION?

MHC class I MHC class II

Expression: all nucleated cells professional APCs

Source of Ag: endogenous exogenous

T cell co-receptor: CD8 CD4

MHC protein: single chain (heavy) two chains ( and )

Salient characteristic: peptide transporter peptide loading comp.

DENDRITIC CELLS INITIATE T CELL RESPONSES BY PRESENTING ANTIGENS

TO NAÏVE T CELLS

Text Figure 8.14

TWO SIGNAL HYPOTHESIS OF NAÏVE T CELL ACTIVATION

(e.g. mature dendritic cell)

TWO SIGNALS ARE REQUIRED FOR A NAÏVE T

CELL TO BECOME ACTIVATED, OTHERWISE

ANERGY (UNRESPONSIVENESS) IS

INDUCED

CONSERVATION OF STRUCTURE IN MHC MOLECULES AND PEPTIDE-BINDING GROOVE

• The 1 and 2 portions of MHC class I and 1 and 1 of MHC class II form mirror images of each other to create the peptide binding groove.

• Despite modest sequence homology, MHC class I and II have evolved to create similar, though not identical binding grooves from a single chain (MHC I) or two chains (MHC II).

STRUCTURE OF MHC MOLECULES AND PEPTIDES BOUND TO THE GROOVE

• MHC class I molecules bind 8-10 amino acid peptides whereas MHC class II bind 12 or longer peptides.

• MHC class II more promiscuous about length of peptide bound.

Mouse I-Ak

Human HLA-DR3

Mouse H2Kb

STRUCTURE OF MHC MOLECULES AND PEPTIDES BOUND TO THE GROOVE

MHC class I + peptide

MHC class II + peptide

MHC CLASS II ANTIGEN PROCESSING• MHC class II molecules present antigens taken up by the cell through endocytosis.• MHC polypeptides ( and ) are synthesized on ER and are chaperoned to a specialized

antigen loading compartment by invariant chain (Ii); invariant chain serves two purposes, it functions as a chaperone and occupies the peptide binding groove to stabilize MHC class II and prevent other peptides (self) from binding until MHC arrives in the loading compartment.

• Invariant chain is cleaved by acid proteases until it leaves only the peptide-binding portion (CLIP) in MHC.

• Endocytosed antigens are also cleaved by acid proteases; low pH requirement for proteases to become activated.

• HLA-DM catalyzes the removal of CLIP and binding of antigenic peptide.

Text Figure 5.10pH 7

pH 5

pH 5

pH 5

THE ROLE OF PROTEOLYSIS IN MHC CLASS II PRESENTATION

• Proteolysis for MHC class II antigen presentation occurs in endocytic vesicles by proteases that are active at low pH.

• Cysteine proteases such as the cathepsins degrade proteins into short peptides for MHC class II presentation.

• Invariant chain is also degraded by cathepsins into CLIP.

THE ROLE OF CATHEPSINS IN MHC CLASS II ANTIGEN PRESENTATION

• Cathepsin L is especially important in thymic epithelial cells, which present antigen for positive selection of thymocytes.

Nakagawa et. al., Immunity 10:207, 1999

• Cathepsin S seems to be especially important in B cells and dendritic cells, less so for macrophages.

Nakagawa et. al., Science 280:450, 1998

I-Ab Ii CLIP

ROLE OF HLA-DM IN THE MHC CLASS II PATHWAY

• HLA-DM facilitates the removal of CLIP and binding of antigenic peptide into the MHC class II binding groove.

• HLA-DM activity is pH-dependent.

pHFluorescence measurement

MHC class II

11

2 2Anti-MHC in microtiter plate

11

2 2

HLA-DM or control protein

Protein concentration

Sloan, et. al., Nature 375:802, 1995

Biotinylated peptide

Streptavidin fluorescent tag

lysosome

MHC class II

MHC CLASS II PROTEINS ARE LOADED WITH PEPTIDE IN A SPECIALIZED

ENDOCYTIC COMPARTMENT (MIIC)

• MHC class II proteins are enriched in multilamellar vesicles called MIICs (MHC class II compartments) that are distinct from other intracellular vesicles.

Text Figure 5.9

MHC class II

Late endosome

Lysosomes

ER

Golgi

Plasma membrane

Early endosome

0

2

4

6

8

10

12

0 5 10 15 20

Fraction number

Amount (arbitrary units)

Tulp et. al., Nature 369:120, 1994

particle

MECHANISMS OF IMMUNE EVASION (MHC CLASS II)

• Mycobacteria prevent acidification of endosomes.

• Inhibition of acidification will prevent proteases from being activated.

• Without proteases, mycobacterial proteins won’t be processed or loaded into MHC class II.

Sturgill-Koszycki, et. al., Science 263:678, 1994

Mycobacteria

Control

Leishmania

Control

pH

HOW DO NAÏVE CD8 T CELLS BECOME ACTIVATED WHEN THE INFECTED CELL IS NOT

A PROFESSIONAL APC?

BONE MARROW CHIMERAS

• Used to determine the function of certain immune cells of one background against the non-immune cells of another background. Irradiate recipient mouse (kills hematopoietic cells from which immune cells are derived) and inject bone marrow cells from donor mouse. In this way, you populate a mouse of one genetic background with the immune system from a mouse of another genetic background.

CROSS PRESENTATION

• CTL immunity to virus-infected non-hematopoietic cells requires presentation of exogenous antigen. Question addressed: Do non-hematopoietic cells infected with virus activate CTLs to kill infected cells or do hematopoietic cells take up exogenous peptides from environment, present them to T cells?

Polio-OVA

Hematopoietic cell (donor)

gPVR

gPVR-expressing cell (recipient)

Peptides

Sigal, et. al., Nature 398:77-80, 1999.

CONTROL OF CROSS PRESENTATION BY THE MHC CLASS I CYTOPLASMIC DOMAIN

Lizee, et. al., Nature Immunol 4:1065-1073, 2003.

Transfected fibroblasts/CTL Transgenic mice

ER-PHAGOSOME FUSION CREATES A CROSS PRESENTATION COMPARTMENT

Guermonprez, et. al., Nature 425:397-402, 2003

OVA

TAP2MHC class I

CROSS PRESENTATION PATHWAY

Houde, et. al., Nature 425:402-406, 2003.

THE POWER OF MHC TETRAMERS

• MHC heavy chains are engineered with linker to create tetramer.

• Peptide is added in solution or engineered to covalently bind to MHC binding groove.

• HLA-A2 tetramers are labeled with a fluorescent tag that allows one to use flow cytometry to determine the frequency of antigen-reactive T cells; previously this was evaluated by limiting dilution analysis.

Altman, et. al., Science 274:94, 1996

HLA AND DISEASE ASSOCIATION

• Some diseases are associated with specific MHC alleles, though the role that MHC plays in the disease isn’t clear.

CD4 T-CELL

TCR

MHCCLASS II

CD4

CD3

CD8 T-CELL

ANTIGEN PRESENTING CELL

TCR

MHCCLASS I

CD8

CD3

T-CELL (ALL

PHENOTYPES)

TCR

CD1

CD3

MOLECULES OF T-LYMPHOCYTE RECOGNITION

9 aa peptide

2m 2m

15 aa peptide

lipid

DIFFERENT CD1 MOLECULES TRAFFIC TO DISTINCT INTRACELLULAR LOCATIONS IN

HUMAN DC

Sugita, et alTraffic 2000

CD1 LAMP CD1+LAMP

CD1a

CD1b

CD1c

Sugita, et al. Immunity

2000

MYCOBACTERIAL ANTIGENS THAT ACTIVATE CD1-RESTRICTED T CELLS

Mannosyl-1-phosphoisoprenoid

Glucose monomycolate

Mycolic acid

Phosphatidylinositol mannoside

CD1b antigens CD1c antigenCD1a antigen

Mycobactin

EXAMPLES OF MHC II EPITOPES THAT REQUIRE PROCESSING IN DISTINCT

SUBCELLULAR COMPARTMENTS

Antigen: Early Endosome: MIIC:

HEL (Zhong et al. 1997)

46-61 -

35-45 -

116-129 -

S. pyogenes M5 (Delvig et al. 1998)

17-31 -

308-319 -

Influenza Virus (Pinet et al. 1998)

HA H3 307-318 -

M1 18-29 -

CD4+T-CELL 1

TRANSLATIONAL THERAPIES TARGETING

MHC ANTIGEN PRESENTATION PATHWAYS

EARLY ENDOSOME

TCR

CD4LATE ENDOSOME/LYSOSOME/M II C

GOLGI

Ag

CD4+T-CELL 2TCR

?MHC II

?

?

M II C

Classical Pathway

Alternative Pathway

TRANSLATIONAL THERAPIES TARGETING MHC ANTIGEN PRESENTATION PATHWAYS

Leader LAMP-1 Ag

Immunize with DNA

Measure immunological functions

Challenge with microbial pathogen and measure survival

CD4

IFN

-

CD4

IFN

-

Unimmunized Immunized

0

20

40

60

80

100

120

0 5 10 15 20 25

Time (weeks)

% survival

Immunized

Unimmunized

CD8+ T-CELL

CD4+T-CELL

PROTEOSOME

MHC CLASS I PATHWAY

MHC CLASS II PATHWAY

pH<5

ENDOSOME

TAP

MHC CLASS I

TCR

TCR

CD8

CD4

M II CER

MHC CLASS II

GOLGI

Ag

Ag