Transcript B cell

Chapter 4 Antigens and Antibodies
Ag
Ab
Complementarity of interacting surfaces of Ab and Ag
Oct 17, 19 & 24, 2006
你需要學習的課題:
1.「好」的免疫原 (immunogen) 有些什麼特質？
2. 什麼叫做抗原決定區 (epitope)？
3. B-cell epitope 有何特性？
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4. 抗體分子的基本構造及各部位的名稱。
5. 抗體的種類、特性及功能。
6. 單株抗體與多株抗體。
Outline
1. Immunogenicity versus antigenicity
2. Epitopes
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3. Basic structure of antibodies (Abs)
4. Ab-binding site
5. Ab-mediated effector functions
6. Ab classes and biological activities
7. Antigenic determinants on immunoglobulins (Ig)
8. The B-cell receptor
9. The Ig superfamily
10. Monoclonal Abs
Immunogenicity vs. Antigenicity
Immunogenicity 免疫性:
the ability to induce an Ab and/or
cell-mediated immune response
Antigenicity 抗原性:
the ability to combine specifically with
Ab and/or cell-surface receptors (Ig/TCR)
- Although a substance that induces a specific
immune response is usually called an antigen,
it is more appropriately called an immunogen.
- Although all molecules that have the property
of immunogenicity also have the property of
antigenicity, the reverse is not true.
- Some small molecules, called haptens, are
antigenic but incapable, by themselves, of
inducing a specific immune response. In other
words, they lack immunogenicity.
DNP: dinitrophenol
(bovine serum albumin)
{
Landsteiner’s work demonstrated both the specificity of the
immune system for small structural variations on haptens and
the enormous diversity of epitopes that the immune system is
capable of recognizing.
Factors That Influence Immunogenicity
Intrinsic properties of an immunogen:
- Foreignness
- Molecular size
- Chemical composition and heterogeneity
- Susceptibility to antigen processing and presentation
The biological system:
- Genotype of the recipient animal
- Immunogen dosage and route of administration
- Adjuvants
Foreignness
- Generally, the greater the phylogenetic
distance between two species, the greater the
structural (and therefore the antigenic)
disparity between them.
- Some macromolecules (e.g., collagen and
cytochrome c) were highly conserved
throughout evolution and therefore display
very little immunogenicity across diverse
species lines.
- Conversely, some self-components (e.g.,
corneal tissue and sperm) are effectively
sequestered from the immune system, so
that if these tissues are injected even into
the animal from which they originated,
they will function as immunogens.
Molecular size
- There is a correlation between the size
of a macromolecule and its immunogenicity.
- The best immunogens tend to have a
molecular mass >100,000 daltons (Da).
- Generally, substances with a molecular
mass less than 5,000 – 10,000 Da are
poor immunogens.
Chemical composition
and complexity
- Synthetic homopolymers tend to lack
immunogenicity regardless of their size.
- All 4 levels of protein organization –
primary, secondary, tertiary and quaternary –
contribute to the structural complexity of a
protein and hence affect its immunogenicity.
Four levels of protein
organizational structure
For Ab (B cell) responses:
Proteins are the most potent immunogens, with
polysaccharides ranking second. Lipids and nucleic
acids of an infectious agent generally do not serve as
immunogens unless they are complexed with proteins
or polysaccharides.
For T cell responses:
Only proteins and some lipids (glycolipids and
phospholipids) serve as immunogens.
Susceptibility to antigen
processing and presentation
- The development of both Ab-mediated and
T-cell-mediated immune responses requires
interaction of T cells with Ag that has been
processed and presented together with MHC
molecules.
- Large, insoluble macromolecules generally
are more immunogenic than small, soluble
ones because the larger molecules are more
readily phagocytosed and processed.
- Molecules that cannot be degraded (e.g.,
polymers of D-amino acids) and/or cannot
be presented with MHC molecules are
poor immunogens.
The biological system contributes
to immunogenicity
- Genotype of the recipient animal
- Immunogen dosage and route of administration
- Adjuvants
Immunogen dosage and route of
administration
Doses: too low  no response, too high  tolerance
Exposure: repeated administration (booster) over a period
of time is usually more effective
Routes: orally (從口入的)
parenterally (非從口入的)
- intravenous (iv) : into a vein
- intradermal (id) : into the skin
- subcutaneous (sc) : beneath the skin
- intramuscular (im) : into a muscle
- intraperitoneal (ip) : into the peritoneal cavity
為什麼 Ag 的量、接觸次數 及 路徑
與免疫反應的強度有關？
Adjuvants 佐劑
- Latin adjuvare, to help
- Substances that, when mixed with an
antigen and injected with it, enhance
the immunogenicity of that antigen.
Effects of adjuvants
1. antigen persistence is prolonged
- slower release of antigen at the injection site
e.g., alum 明礬 [AlK(SO4)2],
2. costimulatory signals (p. 159) are enhanced
- increased expression of B7 molecules on APC
 maximal activation of TH cells
3. local inflammation is increased
4. nonspecific proliferation of lymphocytes is stimulated
- formation of a dense, macrophage-rich mass of cells
called a granuloma 肉芽腫
e.g., incomplete Freund’s adjuvant (IFA),
complete Freund’s adjuvant (CFA)
1. 在實驗動物中製備抗體時，經常使用 CFA
(complete Freund’s adjuvant)，其作用機轉
為何？
2. 為何局部發炎反應能增強 Ab 反應？
Epitopes
- Lymphocytes do not interact with, or recognize,
entire immunogen molecules; instead, they
recognize discrete sites on the macromolecule
called epitopes, or antigenic determinants.
- epitopes: immunologically active regions of an
immunogen, that bind to Ag-specific
membrane receptors on lymphocytes
or to secreted Abs.
T-cell and B-cell epitopes
- The recognition of antigens by T cells and B cells
is fundamentally different.
- Because B cells bind antigen that is free in
solution, the epitopes they recognize tend to be
highly accessible sites on the exposed surface
of the immunogen.
- T-cell epitopes are peptides combined with MHC
molecules. Thus, there is no requirement for
solution accessibility such as B-cell epitope.
Properties of B-cell epitopes
1. B-cell epitopes on native proteins generally
are composed of hydrophilic a.a. on the
protein surface that are topographically
accessible to membrane-bound or free Ab.
2. B-cell epitopes may be composed of
sequential or nonsequential amino acids.
Sperm whale myoglobulin contains 5
sequential B-cell epitopes
Hen egg-white lysozyme (HEL) composes
one nonsequential (conformational) epitope
Contact with Ab light chain
Contact with Ab heavy and
light chains
Contact with Ab heavy chain
Ab to native HEL does not bind to reduced HEL
3. B-cell epitopes tend to be located in flexible regions
of an immunogen and display site mobility.
- site mobility of epitopes maximizes complementarity
with the Ab’s binding site
4. Complex proteins contain multiple overlapping
B-cell epitopes, some of which are immunodominant.
- Most of the surface of a globular protein is potentially
immunogenic.
- Some epitopes, called immunodominant, induce a more
pronounced immune response in a particular animal than
other epitopes.
Basic structure of Abs
Electrophoresis of immune serum
(Tiselius & Kabat, 1939)
Immune sera
after reaction
with Ag
g- globulin (gG)
Immunoglobulin (Ig):
IgG, IgM, IgA, IgE, IgD
Antibody (Ab)
Ab molecules contain 4 peptide chains
- A dimer of heterodimers
V: variable
C: constant
m : IgM
g :IgG
a :IgA
d : IgD
e : IgE
(H)
(L)
100 kDa
150 kDa
→ numerous small peptides
Fab fragment:
antigen binding
45 kDa
22 kDa
Fc fragment:
crystallizable
50 kDa
50-55 kDa
Antibody to the Fab fragment could react with both the H
and L chains, whereas antibody to the Fc fragment reacted
only with the H chain.
 Fab consists of portions of an H and a L chain.
 Fc contains only H chain components.
- A heterogeneous spectrum of antibodies
in the serum g-globulin fraction
- Multiple myeloma: a cancer of Abproducing plasma cells
- Myeloma protein: 95% of the serum Ig
- Bence-Jones proteins: the excess light chains
in the urine.
- MOPC: mineral-oil induced plasmacytoma
in mice
Heavy and light chains
are folded into “domains”
(IgG, IgD, IgA)
(IgM, IgE)
Associations between domains of an Ab molecule
2 Ag-binding sites:
Immunoglobulin Domains
- each contains about 110 a.a. residues and an
intrachain disulfide bond that forms a loop of 60 a.a.
Variable-Region Domains
- hypervariable regions:
(15% - 20% of the variable domain)
= complementarity-determining regions (CDR)
CDR1, CDR2, CDR3
- framework regions (FR)
- diversity in the VH domain is concentrated in CDRs
Variability of a.a. residues in the VH and VL domains
CDRs bind Ags
number of different amino acids at a given position
Variability = _____________________________________________________
frequency of the most common amino acid at a given position
Conformational changes may be induced
by antigen binding
─ : after binding to the Ag
CDRs of L chain : L1, L2, L3
CDRs of H chain : H1, H2, H3
─ : before binding to the Ag
Fab
Ab-mediated effector functions
- Antibodies generally do not kill or remove
pathogens solely by binding to them.
- While V regions bind to Ag, the CH region
is responsible for a variety of collaborative
interactions with other proteins, cells, and
tissues that result in the effector functions
of the Ab responses.
Ab-Mediated Effector Functions
- Opsonization (調理作用) is promoted by Ab
- Abs activate complement (補體 ) (chapter 7)
- Antibody-dependent cell-mediated cytotoxicity
(ADCC) kills cells (p. 366 chapter 14)
- Some Abs can cross epithelial layers by transcytosis
Ab promotes
opsonization
through a
binding to Fc
receptors (FcR)
on phagocytes
Ab activates
complement
-mediated
cytolysis or
promotes
opsonization
through a
binding to C
receptors on
phagocytes
Antibody-dependent cell-mediated cytotoxicity (ADCC)
(Figure 14-15)
Transcytosis
- movement of Ab across epithelial layer
- delivery of IgA to the mucosal surfaces of the
respiratory, gastrointestinal, and urogenital tracts,
as well as its export to breast milk
- transplacental transport of IgG from mother to fetus
Secretory IgA in Breast Milk
Bind to microbes in baby’s digestive tract and
thereby prevent their attachment to the walls of
the gut and their subsequent passage into the
body’s tissues.
Ab classes and
biological activities
5 major classes of secreted antibody
IgG
- most abundant in serum
- 80% of total serum Ig
- 4 IgG subclasses
4 subclasses of human IgG
-
size of the hinge region
no. & position of the interchain -S-S- bond
IgG1>IgG2>IgG3>IgG4 in serum conc.
90% - 95% homologous in DNA sequences
varied effectiveness in placenta transfer and C activation
IgM Pentamer
- monomer on the membrane
& pentamer in secretion
- 5% - 10% serum Ig
- 1st Ab in neonates
- 1st Ab in primary
response
- more efficient in
agglutination & C fixation
- J (joining) chain is
required for polymerization
of the monomers to form
pentameric IgM
- also present in mucosal
surfaces
IgA Dimer
- 10% - 15% of total serum Ig
- monomers, dimers, trimers and
tetramers in serum
- predominant in external secretions,
e.g., breast milk, saliva, tears, and
mucus of the bronchial, genitourinary,
and digestive tracts
Secretory IgA
Dimers and tetramers in secretion with a secretory component
Formation of Secretory IgA
Transcytosis
IgE
- potent biological activity
- extremely low conc. in serum
- mediates the immediate hypersensitivity reactions
- responsible for the symptoms
of hay fever, asthma, hives, and
anaphylactic shock
Allergen cross-linkage of receptorbound IgE on mast cells
- induces degranulation, causing
release of substances that mediate
allergic manifestations
IgD
- 0.2% of total serum Ig
- together with IgM, is the
major membrane-bound Ig
on mature B cells
- thought to function in the
activation of B cells
- no biological effector
function has been identified
Antigenic determinants on Igs
3 Antigenic Determinants
of Immunoglobulins
Isotypes
Allotypes
Idiotypes
Isotypic Determinants
- constant-region determinants that collectively define
each H-chain class and subclass, and each L-chain
type and subtype within a species
Allotypic Determinants
- differences in amino acids in C regions, which
occur in some, but not all, members of a species
Idiotypic Determinants
- The unique amino acid sequence of the V regions of
a given Ab. In some cases, an idiotype is the actual
antigen-binding site
B-cell Receptor
Fc receptors bound to Fc regions of Abs
neonatal Fc
receptor
Fc receptors - essential for many of the
biological functions of Abs
- movement of Abs across cell membranes, e.g.,
the transfer of IgG from mother to fetus across
the plancenta
- passive acquisition of Ab by many cell types,
including B and T lymphocytes, neutrophils,
mast cells, eosinophils, macrophages, and natural
killer cells (a linker between Ab molecules and
various types of cells)
The Ig superfamily
The Ig superfamily -1
The Ig superfamily -2
Monoclonal Abs
Clonal Selection of B Lymphocytes
Production of monoclonal antibodies (mAb)
Questions
•
How to predict whether a molecule is a “good”
immunogen?
•
What are the differences between B-cell epitopes and Tcell epitopes?
•
Draw a schematic diagram of a typical IgG molecule
and label each of the chains, bonds, regions, sites,
fragments and domains.
•
How does Ab kill or remove pathogens?
•
What is the principal of making monoclonal antibody?