微生物及免疫學

Microbiology and Immunology

Lecture 3: Adap.ve immunity – B cells

Reading materials: Chapter 4 and 5
Janeway’s immunobiology 9th

Chialin Hsu
Ins.tute of Microbiology and Immunology
clhsu@nycu.edu.tw
2826-7113, Biomedical Bldg. R309
Human and Germs (A brief clip from NPR)
 Adap.ve immunity – late responder

Innate response: immediate response, but no memory

AdapCve immunity: late response that provides specificity and memory
Why is it necessary to have adap.ve immunity?
• Diversified receptors to recognize pathogens
• B and T cells recognize pathogens and can disCnguish one microorganism from another
• More focused and forceful response to any pathogens
Innate and adaptive immunity differ in their
strategies for pathogen recognition
Innate immunity Adap.ve immunity
Innate immunity uses a fixed AdapCve immunity uses almost
repertoire of cell-surface receptors infinite number of B cell receptors
and soluble effectors to recognize and T cell receptors to recognize
pathogens pathogens
Germ-line encoded SomaCc gene rearrangement
New variants are generated via Diversity are generated via gene
mutaCons and meioCc recombinaCon
recombinaCon
Recognize shared structures of Each B cell receptor or T cell
pathogens receptor has a binding site for a
different ligand
Non-selecCve expansion Only cells with receptors that
recognize the parCcular pathogen
are selected to divide
 Strengths of adaptive immunity

Adap.ve immunity –

• Generates a strong immune response that precisely

targeted against a parCcular pathogen

• The pathogen-specific B and T cells are retained long aQer

the infecCon has been terminated, provide the
“immunological memory”

• The adapCve immune system can keep up with the

mulCplicity of rapidly evolving microorganisms
Phylogeny showing the gene.c evolu.on of influenza A HA sequences of
subtype H3 since 1968
S.mulated B cells become an.body-secre.ng plasma cells

B cells produce an.bodies that recognize specific pathogens

• B cell expresses membrane-bond immunoglobulin (Ig) of a single anCbody
specificity
• Before meeCng an Ag, the mature B cell only expresses Igs in a membrane-
bound form
• The membrane-bound Ig serves as the cell’s receptor for Ag
• When binding to the pathogen, the B cell is sCmulated to proliferate and
differen4ate into plasma cell
• Plasma cells secrete an4bodies of the same specificity
 What is an immunoglobulin or an.body?

Adap.ve immunity is based on B-cell receptors and T-cell receptors

• B-cell receptors are also known as immunoglobulins, while the soluble form
of immunoglobulins are called an4bodies
• Molecules that are recognized by immunoglobulins or T-cell receptors are
called an4gens
• Immunoglobulins or T-cell receptors have specificity for the anCgens they
bind
 B-cell receptor (immunoglobulin, Ig)

• Immunoglobulins (Ig) are made from two different polypepCdes called the heavy
and light chains
• Each “Y” shaped immunoglobulin consists two iden4cal heavy and light chains
• Both heavy chain and light chain have an amino terminal called variable region and
a constant region
• Variable region differs in amino-acid sequence from one Ig to another
• Constant region is very similar in amino-acid sequence between Igs
 Adap.ve immunity – highly specific system

• The differences in the variable regions of Igs create variety of binding

sites that are specific for different anCgens
 An.bodies

• In adapCve immune response, anCbody is a major mean for the body to

clear extracellular pathogens and their toxins

• An individual anCbody can bind to only one (or a very few) anCgen, it is
called specificity

• One person can make up to 1016 (千兆）anCbody repertoire in theory, but

in pracCce, it is limited to 109 （億）(eg. Each person has ~109 B cells)

• An individual B cell produces a unique an4gen specificity, and mature B

cells collec4vely produce many different specifici4es
The structure of the human and mouse Ig classes

What are the differences?

• The lengths of H chain C region
• The presence of hinge region (IgG, IgD, IgA)
• The posiCon of disulfide bonds
• The distribuCon of N-Linked carbohydrate groups

All the Igs occur as monomers in the membrane-bound form, but in the soluble form
IgD, IgE and IgG are monomers, IgA forms dimers, and IgM forms pentamers
The proper.es of Ig isotypes
The immunoglobulin G (IgG) molecule
Igs can be divided into 5 groups: IgA, IgD, IgE, IgG, and
IgM

IgG is the most abundant anCbody in blood and lymph

IgGs are glycoproteins that are built from four

polypepCde chains

Two idenCcal heavy chains (H chains), and two idenCcal

light chains (L chains)

The paired heavy and light chain variable region (V

region) is the an4body binding site

The constant region (C region) have very limited

variaCons
The IgG can be dissected with a protease
DigesCon with the plant protease Papain
can produce three fragments: two arms
and one stem

The arms are called “Fab” (Fragment

AnCgen Binding)

The stem is called “Fc” (Fragment

Crystallizable)

The hinge of the IgG molecule is flexible

so that both Fab arms can bind to the
surface of pathogen Cghtly

The H chain C regions define the five

isotypes of Ig (γ, µ, δ, α, and ε)

The L chain C regions have only two

isotypes: kappa (κ) and lambda (λ)
 Protein domains of the Ig molecules

• The V region of each heavy or light chain is composed of a single variable domain (V
domain):VH in the heavy chain and VL in the light chain

• VH+VL form anCgen-binding site

• The other domains are with lifle diversity and are termed constant domain (C domain)

• Light chain has one C domain CL, heavy chain has 3 to 4 C domain depending on the isotype
and are named CH1, CH2, CH3 or CH4
Hypervariable regions of an.body V domains
• The anCgen-binding site is composed of
three hypervariable regions (HVs)
flanked by less variable framework
regions

• The HVs of the V domain from both

heavy and light chain together form the
hypervariable surface that sit on the 4p
of each Fab arm

• The hypervariable loops are also called

complementarity-determining regions
(CDRs) – because they provide
complementary binding surface to the
anCgen
A brief summary about an.body
An.gen-binding sites vary in shapes and
physical proper.es
• The part of an Ag that can be bound by Ab is called an
an4genic determinant or epitope

• OQen these epitopes are surface molecules of

pathogens, such as carbohydrate or protein

• A macromolecule can contain several different epitopes

• Individual epitopes are usually composed with a cluster

of amino acids or part of polysaccharide chain

• An Ag that contains more than one

epitope (or mulCple copy of the
same epitope) is called mul4valent
Ag
 Polyclonal vs. monoclonal an.bodies

Polyclonal an.-sera Monoclonal an.body

Specificity? Recognize mulCple epitopes of the Single epitope

same anCgen

Affinity? Variable High (can be selected)

Reproducibility? Variable High

Availability? Depends on the animal and its lifeCme Always

Cost? AnCgen and maintenance of animals USD $2,000

Ethics? Need constant monitoring Less concern

Produc.on of monoclonal an.bodies
 Applica.ons of An.bodies

ELISA Western Blot Immunofluorescent microscopy

Monoclonal an.bodies as COVID-19 treatment
 Monoclonal an.bodies as treatment

Why “chimeric” or “humanized” an.body?

• To generate monoclonal Ab, mouse hybridoma is used
• The mouse monoclonal Ab can recognize human Ag with specificity and high affinity,
however, aQer the first treatment, human make Abs against the C region of mouse Ab
(foreign Ag to human!)

To solve the issues:

• Chimeric an4bodies – Keep the mouse-origin of V regions and link to human C regions
• Humanized an4bodies – pick up the CDR loops of the mouse monoclonal Ab and insert into
human Ig
 Genera.on of Ig diversity in B cells –before
mee.ng the Ag

• In all cells, except B cells, the Ig genes are in a fragmented form that cannot be
expressed
• Ig heavy and light chain loci consist of gene segments that are inherited through
germline
• For Ig gene to be expressed, the gene segments must be rearranged to assemble a
funcConal gene
• Ig gene rearrangements occur during B cell development in the bone marrow
 Genera.on of Ig diversity in B cells –before
mee.ng the Ag

• Human Ig genes are found at 3 chromosomal locaCons: H-chain on Chr 14, the κ L-
chain on Chr 2, the λ light-chain on Chr 22
• Different gene segments encode the leader pep4de (L), the V region (V) and the
constant region (C)
• Light chain V region is encoded by 1 V and 1 J, the C region is by 1 C gene
• Heavy chain V region is encoded by 1 V, 1 D, and 1 J segment, the C region is by 1 C
gene
 Random recombina.on of gene segments

• The V, D, and J recombinaCon is called soma4c recombina4on

• A single recombina4on occurs for light chains whereas two
recombina4ons are needed for heavy chains
• The V, D, and J gene segments are selected at random
 The diversity is generated through
combina.ons of mul.ple gene segments

MathemaCcs:
35*5=175
30*4=120
40*23*6=5,520
175+120=295
295*5520=1,628,400

This number is just by the gene

recombina.on, there are even more
tricks to generate diversity!
 The V, D, and J gene segment is flanked by
recombina.on signal sequences (RSSs)

• SomaCc recombinaCon of V, J, and D segments is directed by recombina4on signal

sequence (RSS)
• Two types of RSS – a defined heptamer (CACAGTG) and nonamer (ACAAAAACC) by
a 12 bp spacer
• The other comprises of the heptamer and the nonamer separated by a 23-bp
spacer
• RSS ensures that the gene segments are joined in the correct order
 The recombina.on of gene segments

• The enzyme responsible for recombinaCon binds to 23-bp spacer, and another to 12-bp
spacer, and bring the segments together – the 12/23 rule, to ensure the correct order
• The excised circular DNA has no funcCon and is called the signal joint
• Addi.onal variability can be introduced during the DNA repairing process
 The recombina.on of gene segments

• Enzymes needed to recombine the V, D, and J segments is called the V(D)J

recombinase
• Two of the major component proteins are made only in lymphocytes – recombina4on-
ac4va4ng genes (RAG-1 and RAG-2)
• The other components are present in all nucleated cells, they form RAG complex to
achieve the gene recombinaCon to generate anCbody diversity
• RAG complex can repair double-stranded DNA, bend DNA, or modify the ends of
broken DNA strands
Genera.on of junc.onal diversity during
gene rearrangement
Palindrome: a sequence
that is idenCcal when read
from either end
P nucleo4des

Terminal deoxynucleo.dyl
transferase (TdT),
randomly adds nucleoCdes
N nucleo4des

P nucleo4des + N
nucleo4des = junc4onal
diversity

Have the potenCal to

increase the overall
diversity by a factor of up
to 30,000,000!
 Coexpression of IgD and IgM is regulated by
RNA processing

• CirculaCng B cells that have not yet encounter Ag are called naïve B cells
• Naïve B cells express both IgM and IgD on their surfaces
• Simultaneous expression of both µ and δ chains is accomplished by differen4al
splicing of RNA transcript, not involving DNA rearrangement
 Diversifica.on of Abs aier B cells encounter
Ag – secreted an.bodies

• Binding of Ag to the surface Ig of a naïve B cell triggers the prolifera.on and

differen.a.on, including the secre.on of Abs
• Secreted Abs are produced by an alterna.ve pajern of heavy-chain RNA processing
• Each heavy-chain C gene has two exons – membrane-coding (MC), for the
transmembrane region and the cytoplasmic tail of the isotype; and a secre.on-
coding (SC) sequence encoding the secreted form
Diversifica.on of Abs aier B cells encounter Ag – soma.c
hypermuta.on
• SomaCc hypermutaCon is almost a randomly
introducCon of single-nucleoCde subsCtuCons at a high
rate throughout the rearranged V regions

• This mutaCon occurs at a rate about 1muta4on/V

region/cell division

• 1,000,000 .mes greater than the ordinary mutaCon

rate of a gene

SomaCc hypermutaCon relies on the

enzyme called ac4va4on-induced
cy4dine deaminase (AID)

Only express in prolifera.ng B cells

ResulCng high affinity Abs – affinity

matura4on
Isotype switching: Igs with different C
regions but iden.cal Ag specificity
• Isotype switching (also known
as class switching) is
dependent on AID and
similarly occurs only in B cells
prolifera.ng in response to Ag

• Isotype switching only occurs

during acCve immune
response, and the pajerns of
isotype switching are
regulated by cytokines
secreted by Ag-ac.vated T
cells
Ig isotypes have different physical proper.es
Each human Ig isotype has specialized
func.ons and dis.nct proper.es
Each human Ig isotype has specialized func.ons and
dis.nct proper.es
Summary – Gene rearrangement for B cells
 B lymphocytes

• Lymphocytes include T and B cells. B

lymphocytes are produced in Bone
marrow for mammals. In birds – the
Bursa of Fabricius is where B cells
develop

• B lymphocytes are the producer of

immunoglobulins (Igs)

• More than 60 million new B cells are

generated from bone marrow every day
The life cycle of B cells can be divided into 6 stages

Bone Marrow Secondary Lymphoid Cssues

 B cells develop in the BM and migrate to peripheral
lymphoid organs, where they can be ac.vated by Ags
How each progenitor popula.on progress to B
cell development
• Earliest steps in lymphocyte differenCate begin at the hematopoieCc
stem cell stage and end in generaCon of common lymphoid
progenitor
• Hematopoiesis stages defined by cell-surface markers, transcrip4on
factor expression, and Ig gene rearrangements
– HSCs
• Self-renewing and mulC-potenCal
– MPPs
• Lose capacity for self-renewal, retain mulC-potenCal state
– CLPs
• UpregulaCon B-cell–specific transcripCon factor
• RAG1/2 expression conCnues
• Cells have lost myeloid potenCal, but retain others
– proBs
• The earliest idenCfiable cells of the B-cell lineage
 The site of hematopoiesis

• B-cell development begins in the bone marrow and is completed in periphery

 B-cell development in the bone marrow

• Bone marrow structure is dynamic and complex

– Stem cells within the BM differenCate into many cell types
– Stromal cells in the BM provide support and growth factors to developing cells
 Stages can be defined by Ig rearrangement
and expression

• The progenitor cells have limited capacity for self-renewal

• The major event in the pro-B-cell stage is heavy chain rearrangement
• In large pre-B stage, the heavy chain is fully rearranged and the heavy µ chain is
made
• The light chain rearrangement happens in pre-B-cell stage, first κ light chain the λ
light chain
• When both heavy and light chain have been through successful rearrangement, it
forms membrane-bound IgM
• IgM associates with Igα and Igβ to form funcConal B-cell receptor complex
 B cell development is s.mulated by bone
marrow stromal cells

Non-lymphoid stromal cells provide specialized microenvironments for B cells

• InteracCon of adhesion molecules and their ligands – eg. VLA-4 and VCAM-1
• ProducCon of growth factors such as stem-cell factor (SCF) and IL-7
• Immature B cells leaves the bone marrow and mature in 2nd lymphoid organs
Growth factor for B cell development