Transcript Humoral immune responses “Antibody”

Humoral Responses to Infection
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2015 MICROBIOLOGY &
INFECTIOUS DISEASES
Lecture 10
Ikuo Tsunoda, MD, PhD
Associate Professor
Department of Microbiology and
Immunology
[email protected],
http://tsunodalaboratory.w
eb.fc2.com/
July 22, 2015
Objectives
To understand;
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Structures of immunoglobulins and their
proteolytic digestion
Difference in biologic properties among antibody
classes
VDJ recombination and class switching of
immunoglobulin
Transfer of IgG from mother to fetus (via FcRn)
Mucosal secretion of IgA (via poly-Ig receptor)
Primary and secondary immunoglobulin responses
Effector functions of antibody
Humoral immune responses
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“Antibody” = immunoglobulin is the primary
component of the humoral immunity
“Antigen” = a molecule recognized by
antibody or T cell
“Epitope” is the molecular structure in an
antigen that interacts with antibody or T cell
Proteins (best antigen) >> carbohydrates
(weak) >> lipids, nucleic acids (poor antigen)
Antibodies are composed of polypeptides with both
variable and constant regions
Note:
2 identical heavy chains (green)
2 identical light chains (yellow)
Hinge
The immunoglobulin G (IgG) molecule
Note:
Location of the variable (red)
and constant (blue) regions in
the antibody molecule.
The immunoglobulin constant (C) regions
The constant (C) regions have little variability in
amino acid sequence.
Two different kinds of C regions for light chains :
k
k
kappa (κ)
lambda (λ)
g g
Any of two identical light chains can pair with any two identical
heavy chains to make a complete immunoglobulin molecule.
Five antibody classes (isotypes), determined by the
constant region of the heavy chain
The Structural Organization of the Human Immunoglobulin Classes
Five different kinds of C regions for H chains :
gamma (γ) chains,
mu (µ) chains,
delta (δ) chains,
alpha (α) chains,
epsilon (ε) chains,
IgG
IgM
IgD
IgA
IgE
Each chain is folded into multiple ‘Ig’ domains
Both H and L chains contain multiple loops of around 110 amino acids each.
The heavy chain constant domains (CH) :
The “biological activity” region
Responsible for interactions with other
proteins (eg, complement), cells (eg, mast
cells), and tissues that result in the effector
functions of immunoglobulins
Each chain is folded into multiple ‘Ig’ domains
Both H and L chains contain multiple loops of around 110 amino acids each.
The variable domains (VH, VL) :
The “antigen-binding” region
Together, these domains compose the
sole antigen-binding region of the
immunoglobulin molecule.
The V regions are the first 110 amino acids at the N-terminus of both H and L
chains. This is the region that gives antibody specificity for a given antigen.
Proteolytic digestion of IgG
•Papain treatment produces Fab fragments and Fc fragment
•Fab, antigen-binding; Fc, crystalizable or constant region
•Fc portion interacts with host systems
•Complement activation
•Binding to Fc receptor (FcR) on macrophages and other
cells
Immunoglobulins
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Basic structure
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Heavy and Light chains
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2 identical heavy chains
2 identical light chains
variable (V) and constant (C) regions
disulfide bonds link the chains together
light chains have 2 domains, 1V and 1C
heavy chains have 4-5 domains, 1V and 3-4 C
heavy chains: 5 classes, μ, γ, α, δ, ε
light chains: 2 classes, κ and λ
Membrane bound (mIg) or secreted (sIg)
•Antibody recognizes only a small region (epitope) on
the surface of a large molecule
•Antibody binds an epitope formed by a specific
sequence (linear epitope) or a three-dimensional
structure (conformational epitope)
Antigens can bind in pockets, grooves, extended surface, or
protruding surfaces in the binding sites of antibodies
During B cell development, V, D, and J gene segments
rearrange, creating functional genes that encode
immunoglobulin transcripts.
The process of
rearrangement of
the Ig locus – called
VDJ recombination.
Genetic recombination at the DNA level and processing at the RNA level to
produce functional mRNA
During B cell maturation, V, D, and J gene segments
rearrange, creating functional genes that encode
immunoglobulin transcripts.
For example, a rearranged heavy chain locus is composed of :
• one V gene segment (from 51)
• one D gene segment (from 27)
• one J gene segment (from 6)
variable genes
51
27
V23
D14 J3
constant gene
6
• one C gene segment
*After maturation, the variable gene segment rearrangment
(and therefore the epitope specificity) does not change.
8262 x (200 + 120) = 2.64 x 106
Thus, 2.64 x 106 DIFFERENT specificities are possible from the process of
VDJ recombination alone
8262 x (200 + 120) = 2643840 = 2.64 x 106
A given immunoglobulin has either κ chains or λ chains, never one of each
Note – There are several other forms of antibody
diversification (eg, somatic hypermutation) that
greatly increase the number of distinct antibodies
that the immune system can generate.
Thus, 2.64 x 106 DIFFERENT specificities are possible from the process of
VDJ recombination alone (more than 1011 different specificities in total)
No two B cells are likely to secrete the same Ig (unless
they are clonal progeny), due to the number of different
rearrangements that are possible within the variable
region of the immunoglobulin locus
Murray
textbook, p75
Number of gene
segment, wrong
information:
κ chain: 300 V,
5J
H chain: 3001000 V, 12D, 9J
The Life of a B cell
Naïve B cells die if they do not
see antigen (Ag)
Naïve B cells that encounter
antigen (plus T cell help) :
1. become activated
2. undergo class switching
3. differentiate into either :
plasma cells [antibody (Ab)
secreting]
or
memory cells (non-secreting)
When membranebound Ig (mIg) on the
B cell surface binds
to it’s specific
antigen, the B cell
becomes activated.
Proper activation of the B cell
typically requires contact
with CD4 helper T (TH) cells.
Activated B cells differentiate into :
plasma cells (Ab secreting)
or
memory cells (non-secreting)
Class switching (IgM to IgG, IgE, or
IgA) occurs in response to different
cytokines produced by CD4 helper
T cells
Class switching does not change
the variable region
The usage of heavy chain constant gene segments changes with B
cell activation in a process termed class switching
1
2
3
4
5
but ...... epitope specificity does not change !
Structures of the five major classes of secreted antibody
Important because antibody classes differ with regard to biological properties
IgG and IgA are divided into subclasses
Subclasses differ in the number
and arrangement of the interchain
disulfide bonds.
The principal immunoglobulins in plasma are IgG >> IgA > IgM
13.5 mg/ml
vs.
0.0003 mg/ml
Biological properties of antibody classes differ
Function
IgG IgM IgA IgD IgE
Complement
+
+++
Activation
Opsonization
+
ADCC
+
Placental transport
+
B cell antigen
Naïve
+
+
receptor
Memory
+
+
+
Mucosal secretions
+
Mast cell degranulation
+
Antigen binding
+
+
+
+
+
IgG levels are low from the age of 3 months to 1 year
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IgG is the only isotype to cross the placenta
The transfer of IgG from mother to fetus (placental transfer) is
mediated by an IgG transport protein in the placenta, FcRn,
which binds to the Fc portion of IgG
Infant produces its own IgG at 6 months
Endothelial
cells of fetal
capillary
Villi
Syncytiotrophoblast
Maternal
blood
to the fetal
circulation
FcRn: neonatal Fc receptor for IgG
Syncytiotrophoblasts are bathed in maternal blood and internalize serum
containing maternal IgG. FcRn is expressed in the internal vesicles of the
syncytiotrophoblast. On acidification in the endosome, FcRn binds to
maternal IgG and transcytoses it to the fetal circulation where it is released
at physiological pH.
Dimeric IgA is the predominant in mucosal secretions
The delivery of antibody
to mucosal surfaces
requires their
movement across
epithelial cell layers
Transport is mediated
by the polymeric
immunoglobulin
receptor (pIgR)
IgA in colostrum,
intestinal and
respiratory tracts,
saliva, tears
Some pathogens
produce proteolytic
enzymes that can
cleave IgA
Haemophilus influenzae
(respiratory), Neisseria
gonorrhoeae (genital mucosa)
Secretory
component:
part of the
cleaved pIgR
associated
with the IgA
Immunoglobulin isotypes
are selectively distributed
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IgE is found associated with
mast cells beneath epithelial
surface, including respiratroy
tract, gastrointesitnal tract, and
skin
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IgD exists as membrane IgD,
which serves with IgM as an
antigen receptor on B cells, and
activate B cell growth
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In healthy individuals the Ig
concentration in the central
nervous system (CNS) is low.
The humoral immune response
to infectious pathogens
The primary response to an initial antigen exposure
• long lag phase
• IgM is the first reponder
• gradual switch to IgG
• low affinity antibodies
The secondary response to antigen exposure reflects
immunological memory.
• short lag phase
• very rapid rise in titer
• IgG predominates
• high affinity antibodies
EFFECTOR FUNCTIONS OF ANTIBODIES
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Opsonization – the promotion of phagocytosis of antigens by
macrophages and neutrophils. Protein molecules called Fc receptors
(FcR), which bind the constant region of antibody, are present on the
surfaces of phagocytes.
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Activation of complement – IgM (most effective) and most
subclasses of IgG can activate the classical complement pathway
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Antibody-dependent cell-mediated cytotoxicity (ADCC) – The
linking of antibody bound to target cells (virus infected cells, or some
tumor cells) with FcR of natural killer cells (NK cells), neutrophils,
macrophages, or eosinophils can result in killing of the target cell.
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Neutralization of viruses and bacteria – Prevent attachment to cell
receptors.
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Neutralization of toxins
Antibody-dependent opsonization of bacteria
facilitates phagocytosis of the pathogen
FcγR
IgG
opsonization (op´sŏn-ī-zā´shŭn) The process by which bacteria and other cells are altered in such a
manner that they are more readily and more efficiently engulfed by phagocytes.
opsonin (op´sŏ-nin) Any blood serum protein that binds to antigens, enhancing phagocytosis (e.g., C3b of
the complement system, specific antibodies). [G. opson, boiled meat, opsonin: to prepare for a meal]
Antibody-dependent activation of the complement pathway
Potential outcomes :
- lysis of bacteria via MAC
- induction of inflammatory response
- phagocytosis of bacteria
IgM is the most efficient immunoglobulin for fixing
(binding) complement (Murray p 102)
“Complement fixation” = “Complement activation”
Antibody-dependent cellular (cell-mediated)
cytotoxicity (ADCC) kills the infected target cell
=FcγR
Infection of cells can be blocked by
neutralizing antibodies
Infection of cells can be blocked by neutralizing
antibodies
Antibody can prevent
the attachment of
bacteria to cell surface
Many common
diseases are
caused by
bacterial toxins
Neutralization by IgG antibodies can protect cells from toxins
Neutralization by IgG antibodies can protect cells from toxins
Biological properties of antibody classes differ
Function
IgG IgM IgA IgD IgE
Complement
+
+++
Activation
Opsonization
+
ADCC
+
-+
Placental transport
+
- helminth
B cell antigen
Naïve
+
+
- killing
receptor
Memory
+
+
+
Mucosal secretions
+
Mast cell degranulation
+
Antigen binding
+
+
+
+
+
Murray p73, Table 9-4 is inaccurate
Effector functions of antibodies
http://www.studentconsult.com/content/9780323054706/abbas_sped-up_animations/index.html
https://www.facebook.com/406138889435131/videos/vb.406138889435131/866119870103695/?type=2&theater
Questions (Murray p78)
What is wrong with each of the following statements, and why?
 The laboratory tested a baby for IgM maternal antibodies.
 An investigator attempted to use fluorescent-labeled F(ab')2 fragments
to locate class II major histocompatibility complex molecules on the
cell surface of antigen-presenting cells without cross-linking (binding
two molecules together) these cell surface molecules.
 A patient is diagnosed as having been infected with a specific strain of
influenza A (A/Bangkok/1/79/H3N2) on the basis of the presence of antiinfluenza IgG in serum taken from the patient at the initial visit (within 2
days of symptoms).
 A patient was considered unable to use the complement systems
because of a T-cell deficiency, which precluded the ability to promote
class switching of B cells.
 Analysis of immunoglobulin genes from B cells taken from the patient
described in statement 4 did not contain recombined VDJ variableregion gene sequences.
 A patient was considered to have a B-cell deficiency because serum
levels of IgE and IgD were undetectable despite proper concentrations
of IgG and IgM.
Example of cross-linking by antibody
Anti-HLA (MHC, major
histocompatibility complex)
antibody cross-link two HLA
molecules, activating PI3K
and mTOR
Proteolytic digestion of IgG
Digestion
IgG
no
treatment
pepsin
papain
IgG
F(ab’)2
Fab
Antigen binding/ Cross-linking Fc mediated
Immunostaining /binding two
functions
molecules
ADCC/
together
opsonization
+
+
+
+
+
+
-
-
How to remember papain versus pepsin digestion of IgG?
Papain digestion: pa-pa-in, three syllables = 3 components
Papain
=PAPA(YA) + IN
When IgG molecules are incubated with papain, producing three fragments of
similar size: two Fab fragment and one Fc fragment.
Pepsin digestion: pep-sin, two syllables = 2 components
By Mr. Tameem Islam
Medical Student, LSUHSC
Pepsin digestion produces 1) one F(ab')2 fragment and 2) numerous small
peptides of the Fc portion.
http://www.piercenet.com/browse.cfm?fldID=4E03B016-5056-8A76-4ECA-982DA6CAAC8A
=
=
http://tsunodalaboratory.blog.fc2.com/blog-entry-120.html
Questions (Murray p78)
What is wrong with each of the following statements, and why?
 The laboratory tested a baby for IgM maternal antibodies.
 An investigator attempted to use fluorescent-labeled F(ab')2 fragments
to locate class II major histocompatibility complex molecules on the
cell surface of antigen-presenting cells without cross-linking (binding
two molecules together) these cell surface molecules.
 A patient is diagnosed as having been infected with a specific strain of
influenza A (A/Bangkok/1/79/H3N2) on the basis of the presence of antiinfluenza IgG in serum taken from the patient at the initial visit (within 2
days of symptoms).
 A patient was considered unable to use the complement systems
because of a T-cell deficiency, which precluded the ability to promote
class switching of B cells.
 Analysis of immunoglobulin genes from B cells taken from the patient
described in statement 4 did not contain recombined VDJ variableregion gene sequences.
 A patient was considered to have a B-cell deficiency because serum
levels of IgE and IgD were undetectable despite proper concentrations
of IgG and IgM.
Figure 9-15
Time course of immune responses. The primary response occurs after
a lag period. The immunoglobulin (Ig) M response is the earliest
response. The secondary immune response (anamnestic response)
reaches a higher titer, lasts longer, and consists predominantly of IgG.
Questions (Murray p78)
What is wrong with each of the following statements, and why?
 The laboratory tested a baby for IgM maternal antibodies.
 An investigator attempted to use fluorescent-labeled F(ab')2 fragments
to locate class II major histocompatibility complex molecules on the
cell surface of antigen-presenting cells without cross-linking (binding
two molecules together) these cell surface molecules.
 A patient is diagnosed as having been infected with a specific strain of
influenza A (A/Bangkok/1/79/H3N2) on the basis of the presence of antiinfluenza IgG in serum taken from the patient at the initial visit (within 2
days of symptoms).
 A patient was considered unable to use the complement systems
because of a T-cell deficiency, which precluded the ability to promote
class switching of B cells.
 Analysis of immunoglobulin genes from B cells taken from the patient
described in statement 4 did not contain recombined VDJ variableregion gene sequences.
 A patient was considered to have a B-cell deficiency because serum
levels of IgE and IgD were undetectable despite proper concentrations
of IgG and IgM.
The Life of a B cell
Naïve B cells die if they do not
see antigen (Ag)
IgM, IgD
Naïve B cells that encounter
antigen (plus T cell help) :
1. become activated
2. undergo class switching
3. differentiate into either :
plasma cells [antibody (Ab)
secreting]
or
memory cells (non-secreting)
Questions (Murray p78)
What is wrong with each of the following statements, and why?
 The laboratory tested a baby for IgM maternal antibodies.
 An investigator attempted to use fluorescent-labeled F(ab')2 fragments
to locate class II major histocompatibility complex molecules on the
cell surface of antigen-presenting cells without cross-linking (binding
two molecules together) these cell surface molecules.
 A patient is diagnosed as having been infected with a specific strain of
influenza A (A/Bangkok/1/79/H3N2) on the basis of the presence of antiinfluenza IgG in serum taken from the patient at the initial visit (within 2
days of symptoms).
 A patient was considered unable to use the complement systems
because of a T-cell deficiency, which precluded the ability to promote
class switching of B cells.
 Analysis of immunoglobulin genes from B cells taken from the patient
described in statement 4 did not contain recombined VDJ variableregion gene sequences.
 A patient was considered to have a B-cell deficiency because serum
levels of IgE and IgD were undetectable despite proper concentrations
of IgG and IgM.
The usage of heavy chain constant gene segments changes with B
cell activation in a process termed class switching
1
2
3
4
5
but ...... epitope specificity does not change !
Answers (Murray p78)
What is wrong with each of the following statements, and why?
 The laboratory tested a baby for IgG maternal antibodies.
 An investigator attempted to use fluorescent-labeled Fab fragments to
locate class II major histocompatibility complex molecules on the cell
surface of antigen-presenting cells without cross-linking (binding two
molecules together) these cell surface molecules.
 A patient is diagnosed as having been infected with a specific strain of
influenza A (A/Bangkok/1/79/H3N2) on the basis of the presence of antiinfluenza IgM in serum taken from the patient at the initial visit (within 2
days of symptoms).
 A patient was still considered able to use the complement system,
despite a T-cell deficiency, which precluded the ability to promote
class switching of B cells.
 Analysis of immunoglobulin genes from B cells taken from the patient
described in statement 4 contained recombined VDJ variable-region
gene sequences.
 A patient was not considered to have a B-cell deficiency despite
undetectable serum levels of IgE and IgD, because of proper
concentrations of IgG and IgM.
studentconsult.com
Every B cell expresses a single type of immunoglobulin.
The specificity of that immunoglobulin is determined by
the process of VDJ recombination – rearrangement of
the DNA that encodes the variable region.