8 Pages work
Key points on the assessments:
Try your hand at MCQs, as practice does make you better at them> I have linked in a few on-line MCqs for you to try in the assessment section of the module
Accuracy in the terms used and use of the appropriate terms for appropriate processes is important.
Errors in major facts, will limit your possible marks, such errors include such as calling B cells T cells or vice versa, getting innate and adaptive mechanisms mixed up or confusing MHC classes and T cell co-receptors etc are marked as we read them, not how we think you meant to write it.
Organize your answer so that it has a logical progression. For instance if describing the structure and associated functions of an antibody, start with the core details of the number of proteins involved in making up a generic IgG, the overall structure etc, then drill down into the details of domain structures, bonds, locations of named types of variation etc, class specific differences – of course for antibodies, mentioning the wide range of receptors for antibodies (the Fc receptors) gives context to functionality, as does mentioning the different properties and generalized tissue localizations of the different classes. A diagram is very handy in such cases, providing it is both labelled appropriately and marked as a figure (ie fig 1, fig 2) and then mentioned in the text appropriately (ie see figure 1…)
So above all, answer the question to you fullest ability, without wandering off into irrelevant material or topics…
ASSESSMENT INSTRUCTIONS
Please refer to any further instructions that accompany this document on Moodle
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Figure format(s) accepted (e.g. hand-drawn, composed in Powerpoint, and/or pasted from books/papers) |
Labelled diagrams may be inserted where necessary to support your answer, but should preferably be hand-drawn and the image placed into the document, rather than taken from an existing work. If taken from an existing work, authorship of the image should be acknowledged in the legend to the figure. |
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QUESTION
1. What is the evidence that complement is important in protection against meningococcal disease (40%)? How do meningococci (Neisseria meningitidis) protect themselves from the actions of complement (60%)?
Lymphoid organs
An introduction to the lymphatic system
Dr. Lucy Fairclough
Associate Professor of Immunology
School of Life Sciences
- Describe the role of primary lymphoid organs, specifically in relation to B and T cell development
- Discuss the structure and function of secondary lymphoid organs, specifically B and T cell activation
- Outline the role of the lymphatic system in immunity
Learning Objectives
*
Learning Objectives in Pictures!
*
Cells of the immune system
*
Adaptive Immune Responses
*
Principal lymphoid tissues – primary and secondary
*
Lymphoid organs and development of T and B Cells
*
B cell development in the bone marrow
*
- Precursors in bone marrow
- Self tolerance in immature B cells
- Mature naïve B cell enter circulation and peripheral lymphoid tissues
- Naïve B cells are short-lived – 50 million per day produced in a mouse
- Stimulated B cells (memory cells) are long lived
B cell development in the bone marrow
*
T cell development in the thymus
*
Bone marrow
Thymus
Peripheral lymphoid tissue
T cell development
*
- Precursors in bone marrow
- Maturation, selection of self tolerant cells in thymus
- 200 million short lived cells per day in mice
- Mature T cells enter circulation and peripheral lymphoid tissue
- 1 million mature naïve T cells per day in mice
T cell development in the thymus
*
Secondary lymphoid organs
- Lymph nodes
- Spleen
- Gut-associated lymphoid tissues
*
Lymphoid organs and activation of T and B Cells
*
Secondary lymphoid organs – lymph node
*
Secondary lymphoid organs – lymph node
*
Secondary lymphoid organs – spleen
*
Secondary lymphoid organs – GALT
*
Lymphocyte recirculation
*
The lymphatic system
*
Lymphocytes and Pathogens meet in draining lymph nodes
*
Lymphoid organs
- primary bone marrow, thymus
- secondary lymph nodes, spleen, MALT
Lymphocyte recirculation
- naïve lymph nodes
- memory tissues
Induction of responses
- antigen uptake in tissues
- migration to lymph node – activation of naïve T & B cells
- recirculation of effector T cells to tissues
Summary
Learning Objectives
- Describe the role of primary lymphoid organs, specifically in relation to B and T cell development
- Discuss the structure and function of secondary lymphoid organs, specifically B and T cell activation
- Outline the role of the lymphatic system in immunity
*
Reading List
Immunobiology 7th Edition. Janeway, Travers, Walport and Shlomchik.
Lecture Notes on Immunology 7th Edition. Todd, Spickett and Fairclough
Immunology 7th Edition. Male, Brostoff, Roth and Roitt
The Immune System 3rd Edition. Parham
Roitt’s Essential Immunology 13th Edition.
Complement
Introduction to the complement system and its role in pathogen destruction
Dr. Lucy Fairclough
Associate Professor of Immunology
School of Life Sciences
*
- Describe the key features of complement pathways
- Describe the central role of complement in inflammatory responses
- Illustrate the many functions of complement; cytotoxicity, cellular recruitment and opsonisation
Learning Objectives
*
Innate Immune response
“Recognition”
*
Stages of Complement Action
Complement Pathways
Complement Pathways
Complement Pathways
Physiological Consequences of Complement Activation
The Lectin Pathway
Components of the Lectin Pathway
MASP2
MASP1
Bacteria
Mannose groups
C2
C4
MBL
The Lectin Pathway
Generation of C3-convertase
MBL
C4b
C2a
C4a
C2b
_____
C4b2a is C3 convertase
MASP1
MASP2
Bacteria
Mannose groups
*
The Lectin Pathway
Generation of C5 convertase
MBL
C4b
C2a
C3a
C4a
C2b
MASP1
MASP2
C3b
C4b2a3b is C5 convertase;
it leads into the
Membrane Attack Pathway
*
The Classical Pathway
*
Components of the Classical Pathway
C1 complex C2 C3 C4
C2
C3
C4
*
Initiation of classical pathway
*
Classical Pathway
Generation of C3-convertase
Classical Pathway
Generation of C5-convertase
C4b2a3b is C5-convertase
C4b2a is C3-convertase
The Alternative Pathway
Components of the Alternative Pathway
C3b
B
D
P
Generation of C3 convertase
C3bBb is C3 convertase of alternative pathway
AMPLIFICATION
C3 convertase amplification loop
C3
B
D
P
Membrane Attack Complex
Components of Membrane Attack Complex Pathway
C5
C6
C7
C8
C9
Membrane Attack Complex
Membrane Attack Complex
Membrane Attack Complex
Cytotoxicity
Cellular Recruitment and Activation
Inflammation – C3a and C5a
Cellular Recruitment
Inflammation
*
Opsonization & phagocytosis
Summary
- Describe the key features of complement pathways
- Describe the central role of complement in inflammatory responses
- Illustrate the many functions of complement; cytotoxicity, cellular recruitment and opsonisation
Learning Objectives
*
Recommended Reading
- Immunology, 7th Edition, Male, Brostoff, Roth and Riott, Chapter 4
- Immunobiology, 7th Edition, Murphy, Travers and Walport, Chapter 2
- The Immune System, 3rd Edition, Peter Parham, Chapter 7
*
Antigen recognition and MHC
What you should know by the end of this lecture
The structure of MHC class I and II
The MHC is polymorphic and polygenic
Characteristics of class I and II peptide binding
How molecules are processed and presented via class I and class II MHC
What is the Major histocompatibility complex (MHC)?
Large number of genes which encode class I and class II molecules (called antigen presenting molecules) + associated molecules
Evolved for antigen presentation to T-cells
Class I MHC – presents to CD8+ cytotoxic cells
Cytotoxic cells are there to kill virally infected cells
So MHC is on every cell of our body (as every cell could be infected by a virus)
Class II MHC – presents to CD4+ helper cells
Also on other antigen presenting cells
There are other MHC molecules, but some have different roles in antigen presentation
3
CLASS I MHC
Only 1 alpha chain, with transmembrane region (3 domains)
Peptide binding cleft is part of that alpha chain
Beta 2 microglobulin is a separate molecule which stabilizes structure
Alpha chain is approx. 45kDa, beta-2 macroglobulin is about 12
4
CLASS II MHC
Difference is that there are 2 molecules that form cleft
Beta peptide and an alpha peptide
Alpha chains are approx. 32kDa
Beta chains are approx. 28kDa
5
Chromosome 6p21.3
3.6M base pairs, 200+ genes
Human Leukocyte Antigen (HLA) : Major histocompatibility complex (MHC)
on Chromosome 6, approx 3.6 million bases, approx 200+ genes, half with links to immune function
class I A,B,C
class II DP,DQ, DR
Histocompatibility genes are inherited as a group (haplotype), one from each parent. Thus, MHC genes are codominantly expressed in each individual.
DM – involved in protein/transport to deliver peptides to MHC CII
TAP/LMP – protein processing/generation of short peptides for inclusion into MHC I
Co-dominant expression of MHC alleles
For MHC class IIDQ and DP genes combinations of alpha and beta chains from each chromosome are possible
So expression of multiple MHC on one cell
6 Class I
8+ Class II
Image attribution in presenter notes
Remember the binding groove for class 1 is made up of the alpha 1 & 2 domains of the HLA molecule, whereas in class II , the groove is formed from both the alpha and beta chains
All HLA alleles are expressed, so in terms of classical class 1 and II this is 6 HLA class I (A,B and C from each chromosome) and 6+ class II (DR, DP and DQ) –the variability of class II numbers is due partly to the complexity of the HLA DR: The DR β-chain is encoded by 4 loci, however no more than 3 functional loci are present in a single individual, and no more than two on a single chromosome. Sometimes an individual may only possess 2 copies of the same locus, DRB1*. The HLA-DRB1 locus is ubiquitous and encodes a very large number of functionally variable gene products (HLA-DR1 to HLA-DR17).
The DQA1, DQB1, DPA1, and DPB1 genes are all polymorphic. For DQ and DP, the genetic diversity produced by formation of heterodimers of α and βchains encoded in trans, along with formation of heterodimers encoded in cis, greatly increases the functional diversity of the DQ and DP molecules. In other words, a cell with two different DQA1 alleles and two different DQB1 alleles can be capable of producing four different DQ proteins.
Image: By derivative work: Zionlion77 (talk)MHC_Class_1.svg: User atropos235 on en.wikipediaMHC_Class_2.svg: User atropos235 on en.wikipedia – MHC_Class_1.svgMHC_Class_2.svg, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=5658669
MHC expression on cell types
MHC are both polygenic AND polymorphic
MHC polymorphisms:
HLA Alleles named from 1987-2020
Alleles identified as of 2020
Class I >20,182
Class II > 7,407
Approx 2405 known HLA class II allelic variants (majority are 1512 DRB alleles, 509 DQB1 alleles),
approx 8124 class I alleles
as of March 2020
distribution shows both ethnic and geographically differences
Number of Alleles
This is a slightly older plot taken from Janeway and Travers Immunobiology, it is to indicate that class II allelic variants are largely due to beta chain variants, and Class I variants are fairly equally distributed between the class I A,B and C genes
Even so, some DRB alleles (there are 9 groups) have only a few members, whereas DRB1 variants represent the majority of those found
variation in MHC alleles is focused within the regions forming the binding groove.
for MHC class II, most is found in the beta chain.
The plots depicting this are essentially kabat & Wu variablity plots, complied by examining the amino acid sequence of many allelic variations for the relevant molecule, similary to the way in which variation in immunoglobulin variable domain can be depicted.
Note how the binding grooves and sidewalls of the grooves are sites where many variations are found
Polymorphism
Created by multiple genetic processes
Point mutations
Gene conversion-misalignment in meiosis means copied from a different allele
Gene recombination-Exchange of DNA segments from different chromosomes
Effects of polymorphism in MHC
Variability of peptide expression-allows many thousands of peptides to be presented
Rare that a protein has no peptide that can be bound by MHC
It is important to remember that it is the combination of the peptide-MHC complex is recognised by the TCR
Peptide specificity
Alleles of MHC confer some specificity on the peptide bound
This is particularly in the 2-3 residues that form the ANCHOR sequence
Class I peptides only 8-10aa long
Class II peptides variable length
15
Class I
Class II
Showing peptide in binding groove, Class I can only bind small peptides, ~9aa, whereas the more open-ended binding cleft of class II can accommodate longe peptides ~15mers
16
Anchor residues similar for one allele
But length and position differ between different alleles
Allele A
Allele B
CLASS 1
Anchor residues vary between Alleles of MHC
Anchor residues can vary, but here shown P2 and P9 residues are identified, P9 is often a hydrophobic amino-acid
17
All have same core region but differ in length
Different lengths, but have anchor residues (green) are AA with similar properties
CLASS II
Peptides can vary in size
Peptides that could all bind to a single MHC variant
Providing Anchor residues “fit”, different peptides can be bound by one MHC
Anchor residues are multiple positions, (P1, 4,6 and 9 shown) , more relaxed (permissive) than class I and less easy to define
Differert MHC molecules have different patterns of residues which are conserved.
Here P4 is negatively charged (aspartic Acid (D), other anchor resilues are predominantly hydrophobic
18
How do Peptides get into MHC molecules?
This is very different for class I and class II
Class I binds molecules from host proteins, intracellular viruses and bacteria
Class II can come from intracellular pathogens OR from an extra-cellular source
MHC molecules must have a peptide bound even in the absence of infection- These may be self-peptides
Class I
Intracellular peptides require processing
Antigen processing genes for class 1 are located in MHC
21
Processing of peptides for Class I
LMP and TAP are encoded on MHC locus
Two LMP units form part of the proteosome
Proteosome continuously degrades cytosolic proteins to peptides
Normal process within the cell of recycling
including proteins those from pathogens
expression induced by IFN gamma
Proteosome complex
proteins
peptides
The proteosome is complex of proteins, overall size approx. 2000kDa (roughly), The LMPs (low molecular mass polypeptides) are involved in the processing of proteins into small peptides
LMP2 and 7 are the MHC associated LMPS.
LMPs change the proteolytic specificities of the proteosome.
22
TAP 1 and TAP 2 form a complex that selects peptides
Peptides are then transported into the Endoplasmic reticulum
This is where further processing occurs
Ultimately MHC class I is loaded with peptide
transporters associated with antigen processing (TAP) proteins
23
TAP: transporter associated with antigen processing Are ATP-binding cassette transporters
Calnexin= chaperone protein
ERAAP (mouse)=
ERAP (human) aka: ARTS-1
The class I MHC peptide loading process
Click to edit Master text styles
Second level
Third level
Fourth level
Fifth level
Calreticulin ands ERP57 bind and prevent peptide entry into the binding groove of MHC molecule
This complex binds to the tapasin-Tap complex (the peptide loading complex) Tapasin and ERP57 associate and stabilize the MHC binding groove in an open configuration, facilitiating peptide entry.
ERAP/ERAAP/ARTS-1 – Endoplasmic reticulum aminopeptidase associated with antigen processing : trims peptides that go into MHC class I . ERAAP is important also in that ERAAP-deficient cells are killed by Specific T cells that recognize non-classical MHC complexes
Note there is also a minor, TAP-independent mechanism for loading MHC class I
24
MHC Class II peptide loading
Like MHC class I proteins, class II molecules are synthesized into the rough endoplasmic reticulum
Alpha and beta MHC chains come together, but…
To prevent premature binding of other peptides,
the invariant chain(Ii) binds in the groove.
Ii cytoplasmic tail signals for MHC traffick into
acid endosomes
Invariant chain bound to MHC CII forming a trimer
Ii is also found in the MHC locus
Acid endosomes are around pH 4.
Ii also called CD74, is also found on the surface of macrophages, and is the receptor for macrophage inhibitory factor (MIF)
25
The invariant chain is cleaved in the late endosome by Capthepsin S to leave the CLIP fragment in place
26
CLIP: Class II-associated invariant peptide
Cathepsin S is a lysosomal cysteine protease
Cathepsin L does the same job in thymic cortical epithelial cells
Exogenous antigens (taken in by the cell by phagocytosis) are degraded in lysosome-endosome fusions
MHC-Ii complexes are degraded in acidified endosomes
These all fuse with other vesicles containing HLA-DM.
HLA-DM is a chaperone protein
holds the Class II in an open configuration
Exchanges CLIP for exogenous peptide
Can exchange weak binding peptide for stronger.
MHC class II is released to the cell surface
Loading of MHC Class II with exogenous peptides
27
It is more complex that this, however this excellent review gives more detail than you will need, but is interesting
https://www.nature.com/articles/nri3818
So location of encounter with pexogenous peptide and HLA-Dm govern what types of peptide are loaded into the groove.
Antigen processing and presentation in a B cell
Antigen/pathogen is drawn in attached to the B cell receptor
Antigen is broken down in lysosome-endosome fusions
Peptides can be loaded onto MHC class II as a results
What you should know by the end of this lecture
The structure of MHC class I and II
The MHC is polymorphic and polygenic
Characteristics of class I and II peptide binding
How molecules are processed and presented via class I and class II MHC
Innate and Adaptive Immunity
An introduction to the immune system
Dr. Lucy Fairclough
Associate Professor of Immunology
School of Life Sciences
*
- Consider the threat posed by different classes of pathogens
- Describe the basic features of immunity
- Outline the levels of protection from pathogens
- Briefly describe innate immune responses
- Briefly describe adaptive immune responses
Learning Objectives
*
Classes of Pathogen
*
Classes of Pathogen
*
Classes of Pathogen
*
Requirements of an effective immune system:
Challenge Response
Diverse nature of pathogens Range of defense mechanisms
Vast range of pathogens Vast range of antigen receptors
Rapid growth of microbes Rapid inflammatory response
Minimise damage to host Regulatory mechanisms
*
- RECOGNITION – locate and identify the pathogen
- DEFENSE – repel or destroy the pathogen
An Immune response to infection involves
*
- Specificity
- Memory
- Self-discrimination
Cardinal Features of immune system
*
Levels of defense against pathogens
*
Physical Barriers
*
Physical Barriers
*
Two types of Immune responses
Innate immunity Adaptive immunity
*
Innate Immune response
“Cellular involvement”
*
Innate Immune response
“Cellular involvement”
*
The phagocytic system
*
Innate Immune response
“Recognition”
Pathogen Recognition Receptors (PRRs) and
Pathogen Associated Molecular Patterns (PAMPs)
*
Innate Immune response
“Recognition”
*
Phagocytosis and killing of pathogen
Complement and mast cells
*
Innate Immune Responses
*
Adaptive Immune Responses
*
Adaptive Immune Responses – humoral and cell-mediated
Humoral Immunity
Cell Mediated Immunity
Adaptive Immunity
*
Humoral Immunity
Fab
Fc
*
Immunoglobulin Capabilities
+ ADCC
*
Cell Mediated Immunity
- CD4 helper T cells
- CD8 cytotoxic T cells
*
Adaptive Immune Responses
*
Innate/Adpative Immunity
*
Stages of an immune response
*
*
*
*
In Summary:
*
- Consider the threat posed by different classes of pathogens
- Describe the basic features of immunity
- Outline the levels of protection from pathogens
- Briefly describe innate immune responses
- Briefly describe adaptive immune responses
Learning Objectives
*
Reading List
Lecture Notes on Immunology 7th Edition. Todd, Spickett and Fairclough
Immunobiology 7th Edition. Janeway, Travers, Walport and Shlomchik.
Immunology 7th Edition. Male, Brostoff, Roth and Roitt
The Immune System 3rd Edition. Parham
Roitt’s Essential Immunology 13th Edition.
*
Infection and Immunity
LiFE2080
Antibody diversity
Dr Paddy Tighe (Module Convenor)
Objectives
Outline the types of diversity/location of diversity in antibodies
Outline the process of somatic recombination
Be able to describe what allelic exclusion means
Describe the anatomical sites and consequences of somatic hypermutation
describe central tolerance and its effect on diversity
Outline the mechanism of Class switching
Amino acid variability in immunoglobulins
Isotopic – addresses rare overall identity of an immunoglobulin – which heavy chain class (and subclass where applicable) is used, which light chain (kappa or lambda)
Allotypic – allelic variations across populations – once again associated with both heavy and light chains. Some have effects on the overall physiochemical properties of an antibody, such as altering glycosylation patterns
Idiotypic – identifying the unique structure and sequence that makes a particular antibody ( and applies to TCR as well) different from any other antibody, which is a property associated with the variable regions of the heavy and light chains, and moreover with the CDRs of the variable regions. Niels Jerne defined the idiotype as the set of epitopes (antigenic determinates) on the V region of an antibody.
Amino acid variability in immunoglobulins
Idiotypic variation is focussed at three hypervariable regions (the CDRs) of the variable region gene sequence
Kabat & Wu plots demonstrate this
Kabat and Wu plots represent the variable region mapped as amino-acid positions onto the x-axis (N>C terminal)
`variability on the Y axis is calculated from the number of different amino acids observed at a position DIVIDED by the frequency of the most common amino acid
Light chain CDRs focus around aa 24-34, 50-56 and 89-97
Heavy chain CDRs 31-35, 50-65, 95-102
see http://www.dx.doi.org/10.4049/jimmunol.180.11.7055 and http://www.ncbi.nlm.nih.gov/PMC102431
History
Tonegawa et al Nature 302, p 575 (1983)
Site-specific recombination
Ensuing loss of genomic DNA
Highly specific to immunoglobulin and T cell receptor loci
Highly complex regulation
Lineage specific
Ordered rearrangement of different loci
Regulation of expression from one allele only
Nobel prize 1987 for Physiology and Medicine
the original paper is here: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC431171
Heavy and light Ig chains /TCR genes
Germline sequences are not functional
functional genes are a result of somatic recombination – only occurs in B and T cells
Germline genes are a large cluster of gene segments in a multigene family
Variable region (V) segments
Diversity (D) segments (Ig heavy &TCR beta chains)
Joining (J) segments
Constant (C segments)
TCR
a (14q11.2)
b (7q34)
d (14q11.2)
g (7p14)
Immunoglobulin
H (14q32-33)
k (2p11.2)
l (22q11.2)
Details: IMGT site (http://imgt.cines.fr)
Light chain rearrangement
V & J segments recombine to make the gene encoding the VL domain
V1. . . . . . .V2. . . . . . . . . . . .Vx (100’s) J1 J2 . J3 . . . . Jn
•intervening DNA is lost as circular forms
•A fused V-J segment results
Coding Joint Signal Joint
V1. . . . . . . . .V2J2 . J3 . . . . Jn
+
Choice of V and J segment is random
A specific recombinase system recognises DNA recombination signal sequences (RSS) present at the end of V and start of J regions
Recombination signal sequences
Light chain
Heavy chain
9 23 7
V
J
7 23 9
V
J
7 12 9
9 23 7
D
9 12 7
7 12 9
The 12/23 rule: only a Ig gene segment with a 12 base spacer RSS can be joined to an Ig gene segment with a 23 base spacer
There is a very nice review on the topic of the VDJ recombination process in Nature reviews Immunology 2011 v11, April, p251. it discusses possible mechanisms and influences of chromatin structure and the gene architecture on the process.
http://www/dx.doi.org/10.1038/nri2941
this image is from Immunobiology, 8th edition, chapter 5, figure 5.6
Genes with specific function expressed during B cell development
RAG1 & 2: Proteins Recombination activating genes 1 & 2 – are essential for somatic recombination
Terminal deoxynucleotidyl transferase is a non-templated DNA polymerase which acts on the cleaved DNA ends and adds additional DNA bases
RAG1 and 2 bind to the RSSs, triggers nicking (single stranded cutting of the DNA near the RSS as a key step in somatic recombination
they are expressed in B cell development when heavy and then light chains are recombined.
Terminal deoxynucleotidyl transferase (TdT) is a non-templated DNA polymerase that can act on the cleaved ends of DNA at the recombination site
Recombination complex
Synapsis (requires RAG-1, RAG-2) , HMG 1/2
RAG induced SSB’s are formed
DSB’s are formed (DNA double stranded breaks) + coding end hairpins
Coding ends (CE) are cleaved
Signal joint is precisely made
N and P nucleotide addition occurs @ coding ends
Processing of ends and ligation (XRCC4 and DNA ligase IV)
There is a very nice review on the topic of the VDJ recombination process in Nature reviews Immunology 2011 v11, April, p251. it discusses possible mechanisms and influences of chromatin structure and the gene architecture on the process.
http://www/dx.doi.org/10.1038/nri2941
this image is from Immunobiology, 8th edition, chapter 5, figure 5.6
Problems controlling V(D)J recombination
RAG genes are essentially transposases
Substrate selection errors
End donation errors
>Lymphomas/leukemias
Burkitt’s lymphoma?
( EBV + c-myc (8q24 > 14q32, 2p11 or 22q11)
A progression of rearrangements occurs in the bone marrow
Light chains can rearrange multiple times
(receptor editing)
Receptor editing can rescue B cells which have generated a self-reactive antibody
There is evidence of Germinal centre (ie B cells activated in secondary lymphoid organ) light chain receptor editing as well which might be involved in both tolerance mechanisms and enhancing immune responses to antigens..
There is a detailed article on receptor editing in tolerance and autoimmunity in Annals of the New York Academy of Science,
(2011) 1217 p96-121 ( http://dx.doi.org/10.1111%2Fj.1749-6632.2010.05877.x )
it is estimated that up to 50% of peripheral B cells have undergone receptor editing, though estimates vary widely, reflecting both the types of study and specific methods applied.
there is recent evidence that clonal deletion is more of a selection force that receptor editing for T cells to control autoreactivity.
Joining of Coding Joint DNA is imprecise
V-J, V-D and D-J joining can result in additional nucleotides added to the joint
Non-templated addition by Terminal deoxynucleotidyl transferase (TdT): Termed N region additions
Palindromic additions by template directed fill in by DNA polymerases: Termed P-region additions
Nucleotide additions alter the potential peptide reading frame
N and P region addition
Effects of N and P region additions
Germline
Variable
Adding and removing bases of DNA changes both the number and type of amino acid coded for.
example is sequences of 3 heavy chain VDJ regions, all using the same V, D and J segment, each showing different additions and removal of bases
Allelic exclusion
A single T or B cell will only express the product of a single allele for each of its relevant antigen receptor genes
A B-cell will express a single specificity of IgH chain and a single specificity light chain (k or l)
A single T-cell will express either a unique α and β, or a unique γ and δ TCR pair
This gives monospecificity
Why is mono specificity important?
Number of Igs formed per B cell? if one specificity, then the cell will be specifically activated by encountering an antigen , and produce many clonal specific plasma cells – if lots of different antibody combinations per cell – which one has triggered the cell? all antibodies are made but only a few are specific
Effects on valency – expressing all of the Light and heavy chain genes would give lots of combinations which might/might not be the same on any given antibody molecule so affecting avidity
Allelic exclusion leads to monospecificity of BCR
There is a nice article on allelic exclusion of the IG heavy chain gene reported in J. Immunology, 2014, 192:2460-2470
(http:/www.dx.doi.org/10.4049/jimmunol.1302216 ) which details the involvement of the transcription factor E2A in this process
Somatic hypermutation refines the immune response
In B-cells only
In the secondary lymphoid organs
Targeted mutations in the DNA encoding the rearranged VJ or VDJ segment
altered DNA sequence may encode different amino acids
some mutations will improve binding to antigen.
Occurs simultaneously with class switching
V
J
V
J
D
V
J
V
J
D
Light chains
Heavy chains
Mutation rate peaks over V(D)J junction
• Mutation rate ~ 103 bp/ generation
• (106 x spontaneous rate)
Somatic hypermutation results in small alterations in amino acid sequence within the Variable domains of immunoglobulins
Known details of somatic hypermutation
Mutations : mainly point (4-7% deletions, ~1% duplications)
Not random – hot spot targeted, transitions >transversions
especially serine codons AGC and AGT
CDRs of V gene segments show codon bias
Activation induced cytidine deaminase (AID) is induced
AID is essential for Hypermutation and class switch recombination
Mechanisms for generation of Diversity
choice of multiple genes
random recombination of gene segments
V-J and V-D-J
junctional imprecision
(N region addition, P region addition)
Combination of heavy and light chain proteins
somatic hypermutation
Refining the antibody repertoire:
Central tolerance mechanisms
Peripheral elimination of self reactive cells is important too
Isotype (class) switching
Affects the heavy chain genes only
initially IgD and IgM are made (cell surface)
IgM and IgD isotypes are generated by RNA splicing
Further class switching involves a second specific recombination system
gives a switch to IgG, IgA, IgE
occurs after antigen stimulation
.V2J2 . J3 . . . . Jn. . . . . . . . . . . C? . . . . .C? . . . .
IgM & IgD mRNAs are produced by RNA splicing
DNA (rearranged)
transcription
V1. . . . . . . . .V2J2 . J3 . . . . Jn. . . . . . . . . . . . C? . . . . .C? . . . .
AAAAAAAAA
AAAAAAAAA
• primary transcript
RNA splicing
• messenger RNA
translation
IgM
AAAAAAAAA
IgD
On same cell surface
Recently, IgD was found to bind to basophils/mast cells activating these cells to participate in respiratory immune defense
enzymes involved include activation-induced cytidine deaminase (AID), also needed for somatic hypermutation
what is not shown here in the diagram of the segments is that each heavy chain segment also has a 3’ exon which can be alternatively spliced in after transcription to provide a membrane binding domain for each Ig heavy chain class (this is important for the generation of memory B cells which have membrane bound Ig of the class they have switched to.) thus plasm cells do not splice in this exon, were as memory cells do.
AID is important for Class switch recombination – the introduction of dU (deoxyuracil) into DNA by the action of AID is thought to be the initiating prices to engage the remainder of the machinery required to support class switching.
Summary
Diverse Ig (and TCR) specificities can be generated
Multiple gene segments
Combinatorial rearrangements
Junction alterations
Combinations of different chains (H+L)
Somatic mutation (affinity maturation)
Class switching
Control of the class of antibody response
Central tolerance: Selection of self-compatibility
Peripheral tolerance: Selection of self-compatibility
Infection and Immunity
LIFE2080
B and T cell development and differentiation
Dr Paddy Tighe
What you need to know by the end of the lecture
Where B and T lymphocytes develop
How self-reactive lymphocytes are removed
Positive and negative selection
Peripheral tolerance to self antigens
Development of T & B cells
Need to equip lymphocytes with molecules to respond to antigen (antigen receptors)
Ensure that these receptors do not respond to self antigen (negative selection)
Ensure T cells can respond to to antigens in the context of self-MHC (positive selection)
CD4
TCR
CD8
TCR
CD8+ T-cell
B-cell
CD4+ T-cell
Adaptive response starts with a wide repertoire of possible specificities, for both T and B cells. BUT an essential requirement is to avoid self-reactive antigen receptors, especially on T cells
3
Lymphocyte binding repertoire
Because of the myriad of microbial challenges-antigen binding repertoire has to be extensive –refer to how diversity is achieved from previous lectures
Self-antigen binding must occur during the process of the generation of diversity
There are mechanisms to minimise this because of the devastating consequences (ie autoimmunity)!
Note: we artificially induce tolerance in organ transplantation
Approximately 20 million possibilities of T cell receptor specificy and similar numbers of B cell receptor specificity exist in the average human at any one point in time, and it can change over time (ie is dynamic)
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Tolerance to self
Central Tolerance
Concerns immature T and B cells at the point of development where
B cells have a newly former Immunoglobulin at the surface forming a BCR
Pre-T cells differentiate into CD4+ or CD8+ T cells
Mechanisms
Clonal deletion or inactivation of the cell
A wide range of self antigens are expressed in primary lymphoid tissue to aid this process
Peripheral Tolerance
Relates to mature T cells after they have left the primary lymphoid tissue
Clonal deletion and anergy (lack of responsiveness to stimuli)
Also other mechanisms eg, suppression
Antigens expressed in tissues
B cells develop in bone marrow then migrate to lymphoid tissues
In the bone marrow
Immature B-cells proliferate
Differentiate and develop antigen receptors (surface Ig)
Self-reactive cells are eliminated
Bone marrow stromal cells (ie connective tissues) interact and provide factors (cytokines, inc IL7) necessary for development to eliminate self reactive cells
B-cell
B cells
To be able to function B cells need to:
Encounter antigen
Recognise it
Each naive B-cell only has one specificity of immunoglobulin
Therefore, there is only going to be a few antigens that any one B cell will recognise
Respond to it
Response is driven by support given to the B cell in the secondary lymphoid organs
If all three steps happen, then the B cell differentiates into an effector cell (functional end cell)
recognition
Response/activation
differentiation
A B cell displays many copies of the same specificity immunoglobulin on its surface, each associate with other molecules as part of the B cell receptor
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B cell tolerance
New B cells are produced throughout your life
As gene rearrangement occurs (which defines the antigen specificity) there is a need for mechanism to stop self recognition
This is simple!
If the B cell receptor on the immature B cell detects antigens in the bone marrow it is deleted
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B CELL receptor maturation
Immature B cell expresses surface IgM
While mature, new (termed naïve) B cells expresses surface IgD+ IgM
Developing B cells get signals from stromal cells and once an Immunoglobulin is correctly made and at the surface of the cell, the initial expression is only IgM. Central tolerance is achieved during this stage as self antigens present in the bone marrow and displayed to the new B cells can interact with the new surface IgM, and if the immunoglobulin binds too well, and multivalently (ie perhaps a surface protein expressed many times on a cell), then the B cell will apoptose. Exposure to soluble self-antigens (ie not capable of multimeric binding of Ig leads to anergy of the B cell, or a subsequent failure to response to the same antigenic signal
10
✓
✓
✓
✘
✘
Developmental routes for a B cell
Whilst clonally ignorant cells are weakly self-reactive, the auto-antigen is unable to activate them sufficiently, despite being present.
Note that anergic cells express largely IgD on the surface, and do not respond to antigenic stimulation, so are rapidly lost in competition with responsive B cells with similar antigen specificity.
11
B cell deletion
Immature B cells in BM only express IgM
In order for deletion to occur multivalent ligands have to be bound
Use anti-IgM experimentally – cells die by apoptosis
As a mature B cell is activated by the same ligands – this process only works before they mature
B cell is now expressing IgD on their surface
So is linked in with developmental changes in the B cell.
12
RECEPTOR EDITING
If IgM is bound by antigen in bone marrow immediate elimination (deletion) is NOT the only outcome
The cell an produce a new receptor by editing the existing light chain gene
There is an interval between antigen recognition and apoptosis where cell can be rescued
Receptor editing results in loss of of the old light chain gene and protein with a new one
It is estimated at around 50% of naïve B cells have undergone receptor editing
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INACTIVATION (Anergy)
If the immature B cell encounters monovalent molecule the cell becomes inactivated and unresponsive to stimuli (ANERGIC)
These cells lose their expression of surface IgM
Express mainly IgD, but are released from bone marrow
Anergic cells have a short lifespan due to lack of survival stimuli
Rermember IgM and IgD expression are the result of alternative splicing, so this too is a controllable step.
14
Peripheral Tolerance
There is a need to silence cells in the periphery which encounter self-antigens eg to liver/kidney antigens
Cells can be deleted or anergised (made silent)
As in the bone marrow it depends on valency of antigens
Multivalent (several sites at which attachment to antigen can occur) = able to be deleted in the periphery
Monovalent (one site at which attachment to antigen can occur) = able to be made anergic (silent)
A few self-reactive B cells are OK
Without help, a B cell recognizing antigen is anergised
X
15
Thymus
Bi-lobed organ which sits above the heart
2 compartments-cortex and medulla
Cortex-outer compartment has densely packed immature thymocytes (with high levels of proliferation and cell death) 95-99% will die
Medulla: more sparsely populated-subset of mature thymocytes on the way to being fully matured
In the thymus is a stromal cell network- specialized epithelial cells, dendritic cells and macrophages interacting with thymocytes
Structure of Thymus
mTECs express the AIRE gene, which enables the expression of many, tissue-specific genes in the cells, enabling the broad presentation of self-peptides to developing T-cells, to enable negative selection. Hassal’s corpuscles may have a role in the promotion of dendritic cell function
18
Some thymic epithelial express the AIRE (autoimmune regulator) gene
This enables the AIRE expressing thymic medullary epithelial cells (green) to express self antibens and display them to developing T cells
Red staining is for a Thymic medullary marker.
Mutations in AIRE cause Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED)
19
T-cell precursors migrate from the bone marrow to the thymus
Role of Thymus
Thymectomize- results in a dramatic reduction in T cells
DiGeorges syndrome (humans), nude mice –thymic aplasia (no thymus): do not have T cells
Scid mice have a thymus but a defect in lymphocyte development –do not have T cells
It can be experimentally demonstrated that the thymus is required for T cell development…
Janeway’s Immunobiology 8th ed. Fig 8.17
Normal thymus architecture, but no T cells
NO thymus but normal T cell progenitors
Bone marrow stem cells from nude mouse gives T cell progenitors
Thymic architecture from SCID mouse gives a site for T cell progentitors to develop
So transplanted T cell progenitors can use the normal thymus to develop fully
So existing T cell progenitors can use the transplanted thymus to develop fully
Experimental evidence that the thymus is a key organ of T cell developmentThe spleen is a secondary lymphoid organ and becomes populated with both T and B cells usually, but this only occurs in this model after appropriate thymic development of T cells occurs
Think of it as A house party where the result is the production of mature T cells – SCID mice have the house, but no guests (T cell progenitors), Nude mice have the guests (T cell progenitors), but no house. So transferring guests or house to the relevant lacking mouse enables full development of mature T cells , ie the full house party can occur.
22
T cell development and selection
T cell activation and differentiation
T cell Surface markers
Markers change during development
When progenitor T-cells enter the thymus they lack many later markers
These cells Proliferate and express surface proteins including CD2 but are CD4 and CD8 negative
“double negatives”
Not yet expressed T cell receptor- but this being generated
BOTH CD4 and CD8 are expressed once a functional TCR is present
“double positives” ie are CD4+,CD8+ pre-T cells
At this point selection of the cells for their MHC recognition occurs
Most developing T cells die in the thymus
It is at the double + stage that cells are selected for survival
Most (95%) die by apoptosis
Selected cells go on to become single + CD4 or CD8
Single positives are exported to the periphery from the thymus
Cells express both CD4 and CD8 for a stage of their development, and are called double positive cells at that point
25
The red stained dots indicate where cells are undergoing apoptotic cell death, largely in the Cortex of the Thymus
The large magnification shows the apoptotic cells are surreounded by macrophages (blue)
Most T cells do not survive thymic selection
26
27
The need to recognise self MHC
Ist Phase is positive selection where cells that recognise self MHC peptide complex are selected for survival this happens on double positive cells
If TCR does not recognise self MHC it will die by programmed cell death
This can be illustrated by using mouse models with different MHC backgrounds.
Positive selection
Not only selects for MHC reactive cells
Also co-ordinates the expression of CD4 or CD8 thus determining the type of effector function
Initially expresses both CD4 and CD8
Selection based on whether cell reacts to class I or class II MHC in thymus
Peptides in MHC affect positive selection
Many self peptides are expressed in the thymus due to the action of the AIRE (autoimmune regulator protein)
This ensures developing T cells have TCR which does not react to self-antigens from the rest of the body
But T cells have to have a TCR which will interact with self MHC
Is a delicate balance of affinity.
NEGATIVE SELECTION
If T cell encounters a antigen with high affinity in the thymus it DIES
many self antigens expressed either from circulation or thymus itself
Male antigen in mouse can demonstrate this:
Transgenic mouse with TCR specific for male antigens
In male mice male antigen specific T cells Die
In female mice male antigen specific T cells mature normally
Peripheral Tolerance
Cells once in the periphery may encounter self antigens not presented in the thymus
Need to be able to not react!
Bottom line is need 2 signals, TCR plus costimulation
No second signal =anergy
SUMMARY FOR T CELLS
In thymus cells die unless they receive a positive signal-self MHC/peptide
If they bind to self antigens they receive a negative signal and die
Peripheral tolerance is induced when cells see antigens in the absence of co-stimulation
T cells and MHC
What you should know by the end of this lecture
T cell receptor (TCR) interaction with MHC
Consequences of TCR diversity
TCR function
T-cell effector functions
The T Cell Receptor
In the 1970s T cells were shown to have mono- specificity
Could recognise virally infected cells-but NOT virus alone
Recognition of self+virus
Receptor that does this is the TCR
Self receptor is the MHC
Humans: Approx. 20,000,000 TCR variants at any one time, each one unique
Monospecificity: each unique T cell generated during their development has a unique antigen rececognition for its TCR
Any new T cell will have a unique TCR sequence, but express lots of copies of that one TCR on its surface.
3
The concept of MHC restriction of T cell receptor recognition
This was demonstrated with mouse cells possessing specific mouse MHC (H-2k and H-2d; equivalents of MHC class 1 alleles):
A viral-peptide specific H-2k restricted cytotoxic T cells would kill a cell expressing the correct H-2k variant with TCR-recognized the viral peptide A, as shown here, being presented
It would not however Kill either a H-2k expressing cell with a different viral peptide (B) which that TCR did not recognize, or a H-2d-expressing cell (which the TCR equallty did not recognize, presenting the viral peptide A
4
A simplified version of the concept of MHC restriction.
5
MHC Class I MHC Class II
Each with peptide (yellow) in binding pocket
Images derived from PBD by P.J. Tighe for LNI 7th Ed.
6
A TCR
engaged
with a
MHC class I molecule
T cell
Cell expressing MHC class I
TCR engages with close contact to the MHC-peptide complex two complexes shown, one a’front’ view, one a side view
Orange line is the line chosen for a cross-sectional view in the next slide
7
MHC
TCR
Peptide
Cross section of MHC-peptide-TCR complex
Image: P.J. Tighe, derivatized from PBD structure, for LNI 7th ed.
MHC class 1 alpha chain shown in blue, demonstrating the binding pockets that AA side chains interact with.
Figure also demonstrates both the close interaction of MHC and TCR structures and interaction of TCR with peptide
8
The T cell receptor
Expression of the TCR on the T cell surface requires association with additional proteins
The receptor complex is needed for signalling of TCR engagement with MHC
These proteins are collectively termed CD3
Variation is focussed at the MHC-peptide biding region
Variation is found here
The proteins of the TCR receptor complex (known as CD3, can be targeted by specific antibodies to the external domains so that we can easily stain for T cells (if the antibodies used are fluorescent for example) This marks the cells as T cells.
9
The TCR and the Immunoglobulins share a similar and unique mechanism by which the functional gene is made in each new T or B cell:
SOMATIC RECOMBINATION
TCR
Alpha (Chr. 14q11.2)
Beta (Chr. 7q34)
delta (14q11.2)
gamma (7p14)
Germline organization of the human T-cell receptor alpha and beta loci.
Details: IMGT site (http://www.imgt.org)
Germline genes consist of: Variable (V) segments, Diversity (D) segments, Joining (J) segments and Constant (C) segments
10
Delta and gamma chains are associated with a smaller population of T cells that have a range of functions which are in some ways different to alpha-beta T cells, but are not required reading for the moment.
The L (leader) sequence precedes each V segment, ands is required to allow the TCR to be synthesised into the endoplasmic reticulum
These loci occupy hundreds of thousands of base pairs each.
Alpha chain:
V to J >VJ
Beta chain:
D to J >DJ
Then
V to VJ> VDJ
Note: the germline genes are non-functional
Somatic recombination is lineage dependent:
TCRs in developing T cells, Immunoglobulins in developing B cells
Functional TCR genes are generated by Somatic recombination in the thymus
* Variable DNA sequences occur at the junctions during VJ, DJ & VDJ joining
*
*
*
Even though this picture doesn’t show it well, the VJ and VDJ encoded sections of the proteins for the alpha and beta chains respectively, end up making up the antigen binding site
11
Comparision of TCR and Immunoglobulin rearrangement
Each new (naïve) T cell has a single type of unique TCR
Approx 20 million present, from a potential repertoire of approx. 1×1015~18
12
Changes in immunoglobulin and T-cell receptor genes that occur during B- cell and T-cell development and differentiation.
Those changes that establish immunological diversity are all irreversible, as they involve changes in B-cell or T-cell DNA. Certain changes in the organization of DNA or in its transcription are unique to B cells. Somatic hypermutation has not been observed in functional T-cell receptors. The B-cell-specific processes, such as switch recombination, allow the same variable (V) region to be attached to several functionally distinct heavy- chain C regions, and thereby create functional diversity in an irreversible manner. By contrast, the expression of IgM versus IgD, and of membrane-bound versus secreted forms of all immunoglobulin types, can in principle be reversibly regulated.
T cell Development and relationship with MHC
Details to follow on B and T cell development lecture!
Essentials
Pre-T cells undergo development & gene rearrangement in the thymus
During which time they are
Positively selected for having surface TCR
Positively selected for interacting weakly with MHC+self peptide
Negatively selected interacting either not at all, or too strongly with MHC+self peptide
Survivors exit thymus (~5%)
This is a complex process to be discussed more fully in a later lecture. The final repertoire of T cells which you have circulating in your body is a consequence of this positive and negative selection process.
Interaction of a given T cells TCR with a self MHC (one of your class I or II allelic variants) is essential to allow positive selection, but if too strong, can lead to death of the developing T cell.
This is the process of MHC restriction – your repertoire of T cells is restricted to those T cells whose receptor can interact appropriately with your MHC.
It is also important that a T cell engages both its TCR with MHC and self peptides weakly, and has the correct co-receptor (CD4 or 8) expressed
13
CD4 = T helper cell
CD8 = Cytotoxic T cell
There are two types of co-receptor. Each interacts with
a different MHC class
14
Just to emphasize CD4 and CD8 were first identifiied with monoclonal antibodies that recognised different populations of T cells- long before we knew that their function was to stabilise interactions with particular MHC molecules.
Antibodies to CD4 andCD 8 can be used to identify, and purify specific T cell types
T cell activation and differentiation
Naïve T cells circulate in the blood but enter secondary lymphoid organs eg lymph nodes
T cells mingle with APCs:
If T cell does not encounter an antigen (in MHC) they leave through efferent lymphatics & circulate
If T cell does engage strongly with APC presenting peptide in MHC
proliferate and differentiate into effector cells
T cell activation in the secondary lymphoid organs
Activation retains cells in LN
Activation and interaction leads to:
Proliferation
Differentiation
Differentiated, effector T cells exit the LN
These can be
CD4+ T cells and
CD8+ T cells
16
Some of the mechanisms and signals governing T cell exit from LN are being used as targets for drugs in some autoimmune diseases, to hold T cells back from the circulation
High endothelial venules are specialized and express surface adhesion molecules which naïve T cells interact with. This allows them to them slow down and migrate from the circulation by a process called diapedesis to enter the LN architecture. They are also attracted by a variety of chemokines (CCL21, CXL12
T cell/APC interaction
Initial interactions are with adhesion molecules: ICAM1 (APC) with LFA1 (T cell)
This allows better TCR/MHC/CD4 or 8 engagement
Note – it requires many TCR –MHC engagements to begin the activation process, not just one!
TherE are also additional adhesion molecules which are engaged in this process
17
T cell/APC interaction
Strong binding (ligation) of TCR to MHC-peptide complexes is a first step, but other stimuli are required to activate a T cell
CD4 or CD8 interaction (depending on the T cell and MHC)
Additional co-stimulation aids the stability of the T cell-APC interaction and enables signalling into the T cell
APCs have B7 (aka CD80/86)
Interacts with CD28 on the T cell
APC cytokines complete the initial activation
18
IL2 and activation of T cells
High-affinity IL2 receptor (CD25) is expressed
T cell expresses IL2
Autocrine feedback stimulates cell
Promotes proliferation and differentiation
Differentiation and function
Mature naïve T cells are already committed to being helper (CD4+) or cytotoxic (CD8+)
Cytotoxic cells once activated are effector cells
Helper T cells have a more complex differentiation pathway once stimulated by ag
This is effected by signals from APC
Activation of CD8+ T cells
Very active dendritic cells can directly activate some C8+ T cells
Many however also get additional activation signals though CD4+ T cell help
Engagement to the same APC
increases APC expression of co-stimulatory receptors
IL2 from CD4+ T cell directly acts on CD8+ cell
CD40/CD40L interactions are important in additional co-stimulation of CD4+ T cells and in a different contex, of stimulation fo B cells, which also express CD40
21
Cytotoxic effects CD8+ T cells
CTL recognises MHC Class I peptide complex in secondary lymphoid tissues
Then migrates to tissues and site of infection
These effector cells package cytotoxins into modified lysosomes
On engaging same MHC peptide complex on infected cell…
release this cargo very selectively onto infected cell
CTL: cytotoxic T lymphocyte
22
Two mechanisms of killing:
Both lead to apoptosis of the target cell
23
Apoptosis by these mechanisms far more rapid and controlled than death due to factor withdrawl or other apoptotic stimuli
Perforing opens up pores in the target cell membrane, allowing granzyme entry. Granzyme sets of a pathway of programmed cell death (apoptosis)
FAsL-FAS interactions also set in motion an irreversible pathway of apoptosis
Apoptotic cells are phagocytosed, which in turn supports further presentation of viral antigens on MHC by macrophages.
FAS mediated Killing
24
FAS receptor – member of the TNF family of death receptors
Engagement of the Fas receptor leads to aggregation of intracellular death domains
Recruits procaspase 8
Perforin/Granzyme Killing
Granules contain both Perforin and Granzyme
25
Granules are released from the CTL onto the target cells
Secretion of electron dense granules
Perforin – in vitro can lyse cells – poreformin – but major role is probably to target granzyme to the right place
Granzyme has many targets in the cell (over 300) , but well known targets are the pro-caspases, 8 and 10 which in turn lead to caspases 3 and 7 being activated nd triggering apoptotic death
If a cell has shut down caspase activity – (viruses will do this)
Granzyme can act down stream of caspase –
The immunological synapse, describing the interacting surface between a cytotoxic T cells and a target cell
. TCR-MHC and andehsion molecules are focused at this point of contact, and performing and granzyme containing vesicles concentrated and released specifically here to enable targeted cell killing (Green staining) T cell in blue, target cell in red
The immunological synapse, describing the interacting surface between a cytotoxic T cells and a target cell
. TCR-MHC and andehsion molecules are focused at this point of contact, and performing and granzyme containing vesicles concentrated and released specifically here to enable targeted cell killing (Green staining) T cell in blue, target cell in red
27
Helper T cells
When helper T cell (CD4+) meets APC with relevant antigen
Differentiation options
This depends on a number of factors
Cytokines produced by innate immune sensor cells
Cytokines produced by the interacting APC
Bystander in to LN environment
Cytokines can also inhibit the differentiation of other T cell subsets
29
Cytokines below each T Helper cell subset are indicative of their output capabilities
This also means that Th1 and Th2 produced cytokines can cross-regulate the development of other T cells, this is also true for TGF beta from Tregs which can inhibit Th1 and Th2 development
Functions of CD4+ cells
30
Th1 cell binds to macrophage
Infected with bacteria
And activates the macrophage
To kill
Th1 cells central to killing of intracellular bacteria
Help for Ab production
CD4`+ T cells help B cells to make antibody
Only activate B cells that recognize the same antigen
In secondary lymphoid tissues mature B cells present “their” antigen via MHC class II and pass through T cell zones
When antigen in B cell MHC recognised by Th2 TCR the B cell becomes trapped and activated
B cells encounter antigen in the B cell zones of LN, process and present it
Primary activation signal with BCR engagement,
second signal via CD40 and MHC engagement of TCR
B-cell presents Ag in MHC Class II
it is notable that the peptide in the MHC may be a totally different epitope to that recognised by the ab
Signal 3 through cytokines like IL21 and others
Summary
The TCR binds to a unique peptide within a self MHC
T cells are produced which have this restriction to self-MHC
Activation and differentiation is initiated in secondary lymphoid tissue by APCs
Each type of T cell has a distinct role(s) and all interact with other cells
CD8+ cells kill any infected cell
Th1 cells activate macrophages
Th2 promote recruitment of a variety of innate cells (eosinophils, basophils, Mast cells, and produce IL4, IL5, IL13
TFH cells support B cell activation and differentiation
Th17 cells enhance neutrophil responses
Treg inhibit all CD4+ responses
Cd4+ T cells overall contribute to directing the class of antibody produced by B cells
The different CD4+ t cell subsets alos contribute to directing the humoral response
36
Infection and Immunity
LIFE2080
Antibodies: specificity and function
Dr Paddy Tighe (Module Convenor)
2
Immunoglobulin structure
structure /function relationship
emphasising the binding domains function
Antigen-antibody interactions
Antibody classes
Differences
Effector functions
Summary
Remind of antibody structure
familiar with antibodies as free molecules present in large concentrations in serum and tissue fluids, but is also found on the surface of B lymphocytes – the B cell receptor.
Objectives
Outline the basic structural characteristics of immunoglobulins,
Briefly describe the Immunoglobulin fold and its relationship to antibody structure.
Diagramatically represent the domain structure of an IgG molecule and its relationship to function.
Describe, in brief, the structural details which relate to the specificity of an antibody.
List the main structural and functional differences in different antibody classes.
Associate antibody classes with predominant locations within the body (tissues, fluids, circulation and mucosal surfaces)
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4
Antigens
Antigen: from antibody generator
Many antibodies may bind the same antigen, each a separate site termed an antigenic determinant or epitope
An antigen can also show repeated epitopes
Antigens can be diverse molecular structures
Two key Effector cell types
B lymphocytes (B cells)
make antibodies (immunoglobulins)-cell surface and secreted
Can improve the antibodies made over time
protection against extracellular pathogens & toxins
T lymphocytes (T cells)
express T cell receptors (TCR)
interact with other cells
can kill cells, or facilitate immune responses by other cells
Killer T cells( CD8+), Helper T cells and regulatory T cells (CD4+)
Specific antigen receptors
Immunoglobulin
(antibody)
Size of the adaptive immune system
At any one time, your body may contain up to:
2×107 different specificities of antibodies (ie made by different B cells)
Similar numbers of unique T cell specificities
It changes over time!
20,000,000+ variations on a theme at any one time
out of approx. 150,000,000,000,000 possibilities
You only have about 20-25,000 genes in total:
Just 3 genes given you your antibodies, and 4 genes your T cell receptors
the scope of your adaptive immune system is immense – and it is dynamic, the specificities and abundance of B cells and T cells that are present in your body fluctuates, depending on exposure to pathogens, and age. You have 7 genes (and 2 copies of each) which are enough to generate your entire T and B cell repertoire.
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Immunoglobulins
Serum glycoproteins also found in tissue fluids.
B lymphocytes
surface antigen receptor
(B cell receptor; BCR)
associates with other
cell surface proteins
Plasma cells
produce secretory
immunoglobulin (antibody)
five classes of antibody
IgG, IgA, IgM, IgD & IgE
A B lymphocyte in a secondary lymphoid tissue is activated to proliferate (Clonal expansion and produce more cells, which develop into either memory B cells or more frequently into antibody secreting , short-lived plasma cells (average 2-3 day lifespan) with high antibody output. Memory B cells can survive much, much longer. However there is now evidence of long-lived plasma cells
The Clonal Selection theory
Burnet, FM (1976). “A modification of Jerne’s theory of antibody production using the concept of clonal selection.”. CA: A Cancer Journal for Clinicians. 26 (2): 119–21. doi:10.3322/canjclin.26.2.119. PMID 816431.
the concept of how specific B or T cells are expanded to provide protection against a particular pathogen was proposed in the 1950s by Macfarlane Burnet
the mechanism of somatic recombination was not understood unit the 1976, by Susumu Tonegawa
10
Antibody structure
Enymatic cleavage
Papain
2x Fab, (Fragment- antigen binding)
Fc (Fragment -crystallisable)
• Pepsin
• Fab(2),
• Fc fragments
Papain
Pepsin
understanding of the discrete domains and functions of antibodies came as a result of biochemical work , purifying antibodies and cleaving them with proteases. The terms we often use, the Fab fragment and Fc, came about as a result of these experiments – Fab
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Antibody structure
Bifunctional
2 light chains
(kappa or lambda)
(25KDa)
1.Antigen binding
2. Effector functions
Domain structure
Disulphide linked
2 heavy chains
(subclasses)
(50-77kDa)
there are 9 human heavy chain variants
each has an associated allele
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Ribbon view
Surface view
Top view
Side view
Light chain
Heavy
chain
13
Variable/constant
domain view
Surface view
Top view
Side view
Variable
domains
Constant
domains
Antigen
binding
site
Antibody flexibility
14
The hinge region of IgG1 encompasses amino acids 216-231
IgG2 has a shorter hinge than IgG1,(12 amino acids, four disulfide bridges). The hinge is relatively short and contains a rigid poly-proline double helix, restricting flexibility.
IgG3 has an extended hinge (about four times as long as the IgG1 hinge: 62 amino acids including 21 prolines and 11 cysteines), forming an inflexible poly-proline double helix, the length giving the molecule a greater flexibility.
The hinge region of IgG4 is shorter than that of IgG1 and its flexibility is intermediate between that of IgG1 and IgG2.
15
Serum concentrations and half-life
16
A conserved structure: the “Immunoglobulin fold”
Consider just a light chain
17
Domain structure
Light chain domains
Variable (V)
Constant (C)
Peptide loops (hypervariable regions) are involved in interactions with antigen
18
Hypervariable regions interact with antigen
also called Complementarity determining regions (CDRs)
Heavy and light chains each have 3 CDRs
Amino-acid variation is greatest at the CDRs
19
The combination of H+L CDRs produces the unique antibody combining site
also called the paratope
Antigen is held by non-covalent interactions of the paratope with the antigenic epitope
20
size of antigens and area of interaction varies
21
22
Forces at work
23
“goodness of fit” is the sum of the attractive and replusive interactions between epitope & paratope-
24
Conformational specificity
an antibody may recognise a specific
3D conformation of protein (or other antigen)
linear epitope- primary sequence
…..AlaGlySerProLysGluGlu…..
constrained secondary structure epitope
(eg a loop or turn)
a discontinuous epitope
(tertiary structure epitope)
25
Affinity and Avidity
Affinity
strength of a single antibody(Fab)/antigen bond
Avidity
Binding of a multivalent antibody to a multivalent
antigen
Avidity is likely to be the physiologically relevant affinity
(functional affinity)
Affinity and avidity
26
27
28
Think 3-dimensional when thinking about antibody-antigen interactions
the overall spatial conformation and charge of an epitope is important
Antibody specificity can differentiate between tiny molecular differences
antibody isotypes
30
Ig classes
31
Isotype physical differences
CH1-CH2 hinge length
Interchain disulphide bond location and number
Number of CH domains
Variable glycosylation (Annu. Rev. Immunol. 2007. 25:21–50)
Multimer capability
IgG4half-antibody exchange
Science 14 September 2007: Vol. 317 no. 5844 pp. 1554-1557
32
Multimeric immunoglobulins
33
Tissue distribution of Ig isotypes
Add in IgD’s role in mucosal immunity in upper respiratory tract
Effects of pathogen specific antibodies
Neutralize toxins
Block virus binding to cells
Opsonise pathogens
Activate complement
All aid in remove of pathogen
Antibody receptors (Fc receptors) are present on phagocytic cells
35
Antibody function
Isotypes offer
protection of
different localities
Capabilities :
Neutralisation
Complement
activation
Opsonization
cytotoxicity
36
Fc-receptor bearing cells
37
IgG: location of effector function sites
Ag binding
C4b binding
Fc receptor
binding
C1q fixation
38
Summary
Antibody interactions are via specific domain structures
Immunoglobulin superfamily structures
Antigen interaction via variable domains
Hypervariable loops of H and L chains
Antibody classes provide varied effector functions
HC constant domains provide interaction with complement, fc receptors
Isotypes have different structural and functional properties
bi- or multi valency increased functional affinity and antibody potency
