LECTURE OUTLINE

Nu

ER

MHCI

MHCII

CD8T Cell

TCR

CD4T Cell

Brief overview of immune system to introduce the molecules you will study this semester

The MHC class II antigen processing pathway as an example of how crystals structures yield molecular mechanism

Lisa K. Denzin, Ph.D.Child Health Institute of NJRWJMS/UMDNJdenzinlk@umdnj.edu

MOLECULAR VIEW OF HUMAN ANATOMY - IMMUNE SYSTEM

What is the Immune System?

The immune system is made

up of specialized organs,

tissues, cells and proteins that

keep infectious

microorganisms, such as

pathogenic bacteria, viruses,

and fungi, out of the body, and

to destroy any infectious

microorganisms that invade

the body.

The Immune System is broken down into two parts

Innate Immunity

Response

Speed of Response

Memory

Immediate Slow (>week)

Non-SpecificAntigen Independent

Highly SpecificAntigen Dependent

YesNo

Acquired Immunity

Is always working to protect the body

and does not require any special preparation to stop

infection.

Needs to be 'primed' before it can work to its full effectiveness. Only really effective after it has seen a possible infective

agent before.

Innate versus Acquired (Adaptive) Immunity

Antigen

independent –

How does the innate

immune system

know there is a

problem?

Hallmark of Immune System is Self versus Non-Self (pathogen) discrimination

“Easy” to understand how this works for the adaptive (or antigen-specific) immune response, but how does the innate immune response do this?

Mediated by the expression of Toll Like Receptors (TLRs) on the surface of innate immune cells such as DCs and Macrophages (+others).

These membrane bound receptors recognize structurally conserved molecules (patterns) derived from pathogens (microbes and viruses).

TLR ligation results in immune cell activation and initiation of the immune response – this is the basis of innate immune cell self/non-self discrimination.

http://en.wikipedia.org/wiki/Image:TLR3_structure.png

TLRs are a family of structurally related proteins that recognize different pathogen derived factors.

All have curved lysine rich repeats that mediate specific recognition and activation of the pathway.

TLR Family and diversity

of ligands

http://www.landesbioscience.com/curie/chapter/5350/

Lipopeptides LipopeptidesGlycolipidsLipoproteinsLipoteichoic acidHSP70Beta-glucan

dsRNApoly I:C

LPSHSPsFibrinogenHyaluronic acidNickelOpoids

Flagellin

Lipopeptides

ssRNA ssRNAUnmethylated CpG

TLR Family

ALERTS the innate immune system that something is wrong

Image from www.invivogen.com

Antigen dependent –

How does the innate

immune system

know there is a

problem?

Cells must be “tagged” as infected to activate the adaptive immune system

RIP

“TAG”

or a dendritic cell

The “Tag”

MHC bound to fragments of the antigen (pathogen)

But not known what the interaction of the MHC molecule with the fragment (peptides) looked like.

MajorHistocompatibilityMolecules or HumanLeukocyteAntigens(HLA)

First MHC structureMHC binds peptides – the definitive proof

Bjorkman et. al., (1987) NatureStructure of the human class I histocompatibility antigen, HLA-A2.

Two Types of T cells are generated by two different

classes of Major Histocompatibility Molecules (MHC)

MHC Class I MHC Class II

CD8 T cell CD4 T cell

(T cell receptor) (T cell receptor)

MHC class I – presentation of endogenous antigens (proteins)

http://en.wikipedia.org/wiki/MHC_class_I

MHC class II – presentation of exogenous antigens (proteins)

MHC I and II are structurally similar

MHC I MHC II

Comparison of MHC I & II

MHC I MHC II

Closed peptide binding groveBinds shorter (7-10 aa) peptides

Open peptide binding groveBinds longer (12-24 aa) peptides

Peptide binding by MHC II Molecules

• Peptide always bind in groove with same N-term to C-term orientation

• Ends of groove open to accommodate longer peptides

• Binding energy comes from:

Interactions with side chains of peptide bound into pockets of MHC (also for MHCI)

Interactions (hydrogen bonding) with peptide main chain atoms (less true for MHCI)

N- -C

Peptide binding by MHC molecules

must bind a wide variety of peptides

MHC molecules are unstable when a peptide is not bound

peptides are an integral part of MHC structure

-stable binding prevents peptide exchange at the cell surface

thus, MHC-peptide complexes are good indicators of infection

Stern et al, (1994) Nature

DR1

MHC molecules are highly polymorphic. We all have several different alleles that have unique peptide binding capabilities

MHC IIab

I chain

a b I

ER

NGOLG

I

Proteases(CatS/L; AEP)

abCLIP

The Class II Antigen Processing Pathway

LE/Lyso

CD4 T cell

TCR

abI

APC

Antigen

abI

CLIP solves the problem of an unstable empty (peptide-free) MHC molecule prior to pathogenic peptide binding

CLIP - Class II-associated Invariant Chain Peptides

CLIP binding to MHCII is strange compared to binding of normal antigenic peptides

Peptide binding to MHCII in general is very selective - each peptide usually binds to one MHCIII allele.

CLIP binds pretty well to most MHCII alleles, but binds with a broad range of affinities (mechanism for this is that small aa side chains (Met and Ala) bind in the MHC pockets)

CLIP is a biosynthetic intermediate in the MHCII processing pathway – blocks/preserves the peptide binding groove.

So, how does peptide get in? Perhaps CLIP simply falls out of the groove allowing peptide to bind?

Answer to this question came from the analysis of a group of human MHCII antigen processing mutant cell lines.

Phenotype of these mutants:•class II transport normal

•able to present peptides but not protein antigens

YES!

NO!

MHC IIab

I chain

a b I

ER

NGOLG

I

Proteases(CatS/L; AEP)

abCLIP

LE/Lyso

CD4 T cell

TCR

abI

APC

Antigen

The Class II Antigen Processing PathwayMutant cell lines show

accumulation of cell surface MHC-CLIP

abI

What is missing from the MHCII Antigen Processing Mutants?The Mellins and Pious Labs independently demonstrated the missing factor to be

HLA-DM[Morris et al (1994); Fling et al (1994) Nature]

DM has high homology to MHC class II

Mosyak et al, (1998) Immunity

Class II DM Class II

DM

What is missing from the MHCII Antigen Processing Mutants?

1.Where in the cell is it expressed?

2.Analysis of DM-deficient cell lines. In absence of DM - MHCII is bound to CLIP.

DM itself does not bind peptides or does not bind CLIP and pull it out of the peptide binding groove.

I-Ak

H2-MI-Ak FcRN

[Fremont et al (1998);Mosyak et al (1998) Immunity]

HLA-DM catalyzes CLIP removal from MHCII and promotes peptide binding

DM catalytic activity was only observed at acid pH (similar to that of endosomes)

CLIP

CLI

P r

ele

ase

d f

rom

MH

CII C

LIP c

om

ple

xes + DM

- DM

+ DM

- DM

% M

HC

II-p

epti

de

MHC IIab

I chain

a b I

ER

NGOLG

I

Proteases(CatS/L; AEP)

DMab

abCLIP

DM

The Class II Antigen Processing Pathway

LE/Lyso

CD4 T cell

TCR

abI

APC

Antigen

DM-mediatedPeptide Editing

abI

HLA-DM Edits Peptides and shapes the overall peptide repertoire presented at the cell surface (and seen by T

cells)

DR1 + Peptides (1-6)

-DM +DM

16 hr

Determine PeptidesBound to DR1

Sant et al (2005) Immunological Reviews

[Weber et al (1996) Science; van Ham et al (1996) JEM;Kropshofer et al (1996) EMBO; Lazarski et al (2006) JEM]

How does DM Work - Peptide Editing Function?

Peptides held in MHC class II grove by two different types of intermolecular interactions

1. Co-operative network of hydrogen bonds formed between peptide backbone and side chains of the -helical regions flanking the peptide (sequence independent)

2. Peptide side chains fitting into specificity pockets lining the groove (sequence dependent).

Studies have shown that both sequence independent and dependent interactions are important for DM function/effect.

(CLIP is simply a peptide that must be released or edited out of class II prior to peptide loading)

We just haven’t figured this out yet.

DM-DR1 Crystal Structure

Modeling by Miloje SavicData from Pos et al, (2012) Cell

DM DRDM DR

New Model for DM Function

Binding of the peptide N terminus reverses conformational changes and results in DM dissociation

Pos et al, (2012) Cell

CLIP is bound in DR groove and DR αW43 (red, arrow) stabilizes P1 pocket

Peptide N terminus dissociates from DR groove and DR αW43 rotates away from the P1 pocket, becoming available for interaction with DM. Other DR residues (arrows) move into groove during transition to DM-bound state

DM stabilizes empty DR, and DR αF51 and βF89 protect the hydrophobic P1 pocket

Rapid binding of peptides to partially accessible groove; peptides that do not successfully compete with DR residues (yellow) for P2 site and P1 pocket are not stably bound

HLA-DO / H2-O

HLA-DM HLA-DO HLA-DO + HLA-DM

Class II-like molecule (more similar to classical class II than DM)

Highly conserved across species (human and mouse >80% identical)

Associates with DM in the ER and transported as a DM-DO complex to LE/L compartments

DO Inhibits DM-Mediated Peptide loading

MHC IIab

I chain

a b I

ER

NGOLG

I

Proteases(CatS/L; AEP)

DMab

abCLIP

DM/DO(inactive)

HLA-DO Inhibits HLA-DM resulting in increased cell

surface class II-CLIP

LE/Lyso

CD4 T cell

TCR

abI

APC

Antigen

DOab

DM(active)

abI

DO/H2-O inhibits /alters the peptide-loading function of DM/H2-M

This suggests that perhaps the role of DO/H2-O is to dampen or modulate the class II-bound (self)-peptide repertoire thereby preventing inappropriate T cell activation which can result in autoimmunity

DO/H2-O expression has been shown to:• Modulate autoimmunity• Inhibit (restrict?) immune responses

What is the function of HLA-DO?

DM

DM/DO

DM-DO Crystal StructureDO is an inhibitor – MHC class II mimic

Modeling by Miloje SavicData from Guce et al, (2013) Nature Structure and Mol. Bio.

DM DODM DO

Modeling by Miloje SavicData from Guce et al, (2013) Nature Structure and Mol. Bio. and Pos et al, (2012) Cell

DO

DM

DR

DM

DR

DO looks like MHC II-Substrate mimic of MHCII to inhibit DM Function

DO

T cell-APC Interaction via the TCR

T cell-APC Interaction via the TCR

Structures of the 2C–dEV-8 complex K. C. Garcia (Stanford University)

T cell-APC Interaction via the TCR

CD4TCR co-receptor

Co-Stimulation Molecules

Innate versus Acquired (Adaptive) Immunity

B cells make antibodies specific for pathogenic proteins

(bacterial proteins in this example)

CD4 T

Cell

Antibodies

Antibody Structure

Effector Region – mediate different functions5 main isotypes

IgMIgAIgGIgEIgD

Antibody Isoforms and Function

IgM:Expressed on the surface of B cells (monomer) and in a secreted

form (pentamer) with very high avidity. Eliminates pathogens in the early stages of B cell mediated (humoral) immunity before there is sufficient IgG.

IgG: In its four forms, provides the majority of antibody-based immunity

against invading pathogens. The only antibody capable of crossing the placenta to give passive immunity to the fetus.

IgD: Functions mainly as an antigen receptor on B cells that have not been

exposed to antigens. It has been shown to activate basophils and mast cells to produce antimicrobial factors.

IgE: Binds to allergens and triggers histamine release from mast cells and

basophils, and is involved in allergy. Also protects against parasitic worms

IgA:Found in mucosal areas, such as the gut, respiratory tract and urogenital tract, and

prevents colonization by pathogens. Also found in saliva, tears, and breast milk.

http://en.wikipedia.org/wiki/Antibody

Model of T cell help and B cells

NaïveCD4

DC

Mac B cell

TFH

DMDO

B cell

GCB cell

Memory B cellsPlasma Cells

IgM

IgG

Affinity Maturation (mutations introduced into antibody results in higher affinity Isotype switching – depending upon which cytokines are present, Ig isotype switches (IgM to IgG, etc.) Generates antibodies with different effector functions.

http://www.wearepowershift.org/blogs/building-community-immunity-0

tt

TLRs

MHC

TCRCo-stim

Antibody

BCR(membrane

boundantibody)

Putting it all together: anatomy of the immune response to a virus

For more information:

Text Books:

Janeway’s Immunobiology 8th Edition

(by Ken Murphy)

How the Immune System Works(by Lauren Sompayrac)

Free On-line Resource

The Kahn Academy – https://www.khanacademy.org/science/biology/immunology