LECTURE OUTLINE Nu ER MHCI MHCII CD8 T Cell TCR CD4 T Cell Brief overview of immune system to...
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Transcript of LECTURE OUTLINE Nu ER MHCI MHCII CD8 T Cell TCR CD4 T Cell Brief overview of immune system to...
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/[email protected]
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