lecture outline mhc i ag presentation mhc ii ag ...€¦ · what cells express mhc class ii...

Advanced Course in Basic & Clinical Immunology

February 19, 2018

1

Antigen presentation

Kenneth L. Rock, M.D.

Professor & Chair

Department of Pathology

UMass Medical School

Lecture outline

MHC I Ag presentation

MHC II Ag presentation

Antigen presenting cells (brief)


February 19, 2018

2

CD8 T lymphocyte

How do theyrecognize thathese cells ar

abnormal?

The principal adaptive immune defense against cancersvirally infected cells

MHC class I molecule


February 19, 2018

3

Genes

Virus

ERMHC I

Nucleus

MHC class I antigen presentation

TAP

Parham

The MHC I antigen binding receptor

Parham


February 19, 2018

4

From Kuby MHC class I genes

MHC class I genes are highly polymorphic

Now >10,000 alleles of MHC class I genes identified!!


February 19, 2018

5

Nomenclature

(defined by serology)

(defined by DNA sequence)

From Parham – The Immune System

Where are the polymorphic residues?

Class I & II genes are highly polymorphic

What will the polymorphisms affect?


February 19, 2018

6

Clinical importance of MHC polymorphism

Transplant rejection

Susceptibility to infectious disease (e.g. HIV elite controller)

Susceptibility to autoimmune disease

Responses to vaccines & immunotherapy

GWAS HIV elite controlers

The International HIV Controllers StudyScience 2010

Vol. 330 pp. 1551-1557

MHC I alleles


February 19, 2018

7

How many different MHC I molecules can cells express?

H2N C C N C COOH

R R’

HH O

Common

Unique

Peptide Structure


February 19, 2018

8

Peptides bound to MHC molecules

1 2 3 4 5 6 7 8 9N COOH

Side chains not bound

side chains bound by MHC

moleculeFixed (few) amino acids

Can be any of the 20 amino acids

Therefore, a single MHC I molecule can bind huge numbers of peptides (but not all)

With 6 different MHC I molecules, can “cover” much of the antigenic universe

So, how do we get from proteins to

presented peptides


February 19, 2018

9

Parham

MHC class I pathway utilizes the peptides generated from the normal catabolism of

cytosolic & nuclear proteins

ProteinProteolysis by Proteasomes

Active sites


February 19, 2018

10

0

50

100

150

CP

M X

10-

3

0.3 0.6 1.25 2.5 5 10

Vaccinia - OVA

Inhibitor0

50

100

150

0.3 0.6 1.25 2.5 5 10

Vaccinia - SIINFEKL

APC X 10-4

Inhibitor

Effect of proteasome inhibitors on antigen presentation

MH

C I

antig

en p

rese

ntat

ion

Rock et. Al. 1994. Cell 78: 761-771

Immune system modificationof proteolysis


February 19, 2018

11

ImmunoproteasomesThe picture can't be displayed.

The picture can't be displayed.


Antigen

Immunoproteasome








IFNMHC

1i

immunoproteasome ß-subunits

5i

Non-MHC

2i




Proteasome (constitutive)





5

2

Different cleavages result in different peptides

1

[Also a 5t subunit expressed only in the thymus]

Proc. Natl. Acad. Sci. USA 91: 9212-9217

How important are immunoproteasomes?

1i/2i/5i-/-

Immunoproteasome knock out


February 19, 2018

12

H-2Db H-2Kb

Peptides eluded from MHC I

Wt Immuno-Proteasome-/-

Wt Immuno-Proteasome

50% qualitatively different!

Conclusion

The proteasome is required to generate the majority of presented peptides

The immune system evolved modifications of proteasomes to optimize antigen

presentation.


February 19, 2018

13

How do class I molecules access cytosolic peptides?

ass I molecules access cytosolic peptides? Cytosol

ER

ER membrane

Peptide

1i TAP1 5i TAP2

MHC CHROMOSOME 6

MHC I

TAPCytosol-to-ER peptide transporter


February 19, 2018

14

Other events in the ER

MHC I

Chaperones-calnexin-ERP57

Tapasin

TAP

PeptideRibosomes

To cell surfaceEndoplasmic reticulum

MHC I

Tapasin

Encoded in MHC

Thought to play a role of retaining “empty” MHC I in the ER

Also promotes peptide loading of MHC I molecules


February 19, 2018

15

H2-Kb 8mers (some 9mers)

HLA-A,B, C 9-10mers

Size of peptides bound by MHC I molecules

Does the proteasome generate presented peptides in their final

form?


February 19, 2018

16

What is the size of proteasome products?

Cascio et al., EMBO J. 20:2357-66 (2001)Kisselev et al., J Biol Chem 274:3363-71 (1999)

Too short Too longJust right

MHC binding

Are long peptide fragments precursors for epitopes?

epitopeN COOH

Presented

epitope


February 19, 2018

17

Cleavages to generate MHC I peptides

epitopeN COOH

PresentedOnly

ProteasomeOther

peptidase

Summary of key points:• To generate peptides from expressed genes the

immune system has utilized the normal cellular pathway that degrades proteins

• The initial cleavages are made by proteasomes and especially immunoproteasomes, often making long antigenic precursors.

• Most peptides are generated in the cytoplasm

• The correct C-terminal cleavage is ~only made by proteasomes

• Other peptidases can trim the N-terminus of peptides


February 19, 2018

18

N-terminal trimming of peptides

Much of the trimming occurs in the ER

By the aminopeptidase = ERAP1/ERAAP

(note humans but not mice also have an ERAP2)

Importance of ERAP1 in vivo

ERAP1 “knock out”


February 19, 2018

19

*

**

*

*

LCMV immunodominance hierarchy in ERAP1-/- mice

0

1

2

3

4

5

6

7

8

Per

cent

of T

cel

l res

pons

eERAP1 -/-Wild type

Proc. Natl. Acad. Sci. USA. 103: 9202-7

Humans

ERAP1 polymorphisms linked to autoimmune diseases and immune

responses.


February 19, 2018

20

ERAP1 unique-trims with a molecular ruler

EPITOPE

11 10 9 8

EPITOPE

ER

ERAP1

Nature Immunology, 3: 1169-1176

Leu-AMC

Active site

ERAP1 shows allosteric activationAMC


February 19, 2018

21

Leu-AMC

+Active site

ERAP1 shows allosteric activation

Peptides ≤7 AA AMC

Leu-AMC

+Active site

ERAP1 shows allosteric activation

Peptides ≤7 AA

Leu-AMC

+Active site

Peptides ≥8 AA

AMC


February 19, 2018

22

Crystal Structure

Reorientation of Tyr438


February 19, 2018

23

Crystal structure

trigger

Cytosol trim

cleave

Crystal structure

trigger

Cytosol trim

cleave

X


February 19, 2018

24

Summary

Influences responses

ER Trimming has specificity

N-extended peptides are trimmed in the ER by ERAP1

ERAP1 trims with a molecular ruler

1/2

Source proteins for MHC I presentation

Immunoribosomes?

Turn over of the proteome

Products of protein splicing

Defective proteins (DRIPs)?


February 19, 2018

25

Antigen presentation is a “bell & whistle”

MHC I molecules, tapasin, TAP transporter, immunoproteasomes, ERAP1

are not required for cell viability

Microbial immune evasion

PeptideCMV

CMVHSV

EBVAntigen

Endoplasmic reticulum

TAP

Proteasome

MHC I


February 19, 2018

26

Cancer immune evasion

This will be a barrier to cancer immunotherapy

Many cancers are genetically unstable

Can lose expression of cancer antigens, MHC I, Tapasin, TAP

Variable for precision medicine?

E

Summary of Pathway

ERAP

TAPASIN


February 19, 2018

27

MHC II antigen presentation

Macrophage

Microbe

Phagocytosis

Destroy microbe

Infection

Extracellular microbe

Outcomes of bacterial infection


February 19, 2018

28

The major immune defense against cells infected with bacteria are CD4 T

cells

CD4 Tcell

MHC II sample peptides in endocytic compartments

MHC II molecule

ExtracellularAntigens

Microbe

Endocytic uptake

CD4+TH

Vacuoles


February 19, 2018

29

MHC II peptide-binding receptors

Very similar tertiary structure to MHC I

3 molecules = HLA-DR, DP, DQ

Same regions highly polymorphic

Parham

MHC II monitors peptides from phagosomes (& endocytic compartments)

How are these generated?


February 19, 2018

30

The endocytic compartment is also catabolic.

H+ H+

Lysosome

Inactiveprotease

Antigen

Phagosome

Activeprotease

Proton Pump

H+

Peptides

How do class II molecules get to phagosoms/endosomes to sample

these compartments


February 19, 2018

31

Synthesis & assembly of MHC class II molecules

MHC IIInvariant chain

Ribosomes

Endoplasmic reticulum

Clip region

How is class II prevented from being saturated with peptides in the ER & then get to the right compartments?


February 19, 2018

32

How is class II prevented from being saturated with peptides in the ER & then

get to the right compartments?

Invariant chain

Clip region blocks the groove

Sorting sequence to endosomes

Key points• Invariant chain binds newly synthesized class II in the ER

• Invariant chain blocks peptide binding to class II

• Invariant chain directs class II molecules to endocytic compartments

• Peptides are generated in endosomalcompartments by acid optimal proteases


February 19, 2018

33

How are class II molecules activated in endosomal vesicles?

HLA-DM

H+

Endosomal Vacuole (MIIC)

CLIP

Antigen

To cell surface

DM promotes loading/editing of antigenic peptide within antigen presenting cells

DM MHC II

Affinity


February 19, 2018

34

DM

DM

DR DRpeptide

DM-MHCII interaction(from crystal structure of a trapped complex)

Model for DM-dependent peptide exchange

2


February 19, 2018

35

Key Points• Invariant chain is hydrolyzed by proteases in the endocytic compartment

• A fragment of invariant chain (CLIP) is left in the peptide binding groove

• CLIP is removed by HLA-DM

• Peptides bind to class II molecules (facilitated by HLA-DM)

What cells express MHC class II molecules?


February 19, 2018

36

What cells express MHC class II molecules?

All nucleated cells

EffectorCD8+CTL

Dendritic cell Macrophage B cell

MHC I

EffectorCD4+CTL

MHC II

These are precisely the cells that CD4 T cells need to monitor and activate

B cells & macrophages, DCsAll can take up antigen through endocytosis and

present via the mechanisms we’ve discussed so far.

In addition, some have specializations:

MØ & immature DCs can acquire Ag through phagocytosis (important for microbes & cell Ags)

B cells can capture Ag through their surface antibody.This is important for antigen specific B cells to acquire antigen, process it & present peptides to T helper cells

DCs can transfer eaten antigens into the MHC I pathway (called cross presentation)


February 19, 2018

37

Dendritic cells are key antigen presenting cells (APCs) for initiating T cell responses

Why?

The initial frequency of any anti-viral T cell is very low

AntigenX


February 19, 2018

38

The “where’s Waldo” problem

The “where’s Waldo” problem


February 19, 2018

39

Huge territory

How can this work?


February 19, 2018

40

CCR7CCR7

scoutsFort

dendritic cells Lymph node

“Wild west” strategy

Dendritic Cells- Key APCsAnatomic location – located where the antigens are

(mucosa & all tissues)

Have receptors that sense danger (infection & injury) & stimulate the DC

(pattern recognition & cytokine receptors)

When stimulated, DCs express all the signals needed to activate naïve T cells (Ag, costim., cytokines)

Trafficking: Tissues(CCR7) L. NodesT cells(naïve T cells don’t traffic through tissues)


February 19, 2018

41

Implications for cancer

The cells that initially present cancer antigens (dendritic cells) have immunoproteasomes

In the absence of inflammation, cancer cells may lack immunoproteasomes

Therefore, the T cells that are elicited might not optimally recognize the cancer

END

lecture outline mhc i ag presentation mhc ii ag ...€¦ · what cells express mhc class ii...

Documents