pete sieling phone: 825-6964 email: [email protected] [email protected] office: 52-127...
TRANSCRIPT
• Pete Sieling• Phone: 825-6964• email: [email protected]• 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